Humanized anti-beta7 antagonists and uses therefor

ABSTRACT

The invention provides therapeutic anti-beta7 antibodies, compositions comprising, and methods of using these antibodies.

This is is a continuation application of U.S. patent application Ser.No. 14/454,884 filed Aug. 8, 2014, which is pending, which is acontinuation application of U.S. patent application Ser. No. 13/788,246filed Mar. 7, 2013, which is pending, which is a continuationapplication of U.S. patent application Ser. No. 13/348,709 filed Jan.12, 2012, now U.S. Pat. No. 8,779,100, which is a continuationapplication of U.S. patent application Ser. No. 12/390,730, filed Feb.23, 2009, now U.S. Pat. No. 8,124,082, which is a divisional applicationof U.S. patent application Ser. No. 11/219,121 filed Sep. 2, 2005, nowU.S. Pat. No. 7,528,236, which claims the benefit under 35 U.S.C.§119(e) to U.S. Provisional Application Ser. No. 60/607,377, filed Sep.3, 2004, the entire contents of which is hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates generally to the fields of molecularbiology and growth factor regulation. More specifically, the inventionconcerns modulators of the biological activity of integrins containingthe beta7 subunit, and uses of said modulators.

BACKGROUND

The integrins are α/β heterodimeric cell surface receptors involved innumerous cellular processes from cell adhesion to gene regulation(Hynes, R. O., Cell, 1992, 69:11-25; and Hemler, M. E., Annu. Rev.Immunol., 1990, 8:365-368). Several integrins have been implicated indisease processes and have generated widespread interest as potentialtargets for drug discovery (Sharar, S. R. et al., Springer Semin.Immunopathol., 1995, 16:359-378). In the immune system, integrins areinvolved in leukocyte trafficking, adhesion and infiltration duringinflammatory processes (Nakajima, H. et al., J. Exp. Med., 1994,179:1145-1154). Differential expression of integrins regulates theadhesive properties of cells and different integrins are involved indifferent inflammatory responses. Butcher, E. C. et al., Science, 1996,272:60-66. The beta7 integrins (i.e. alpha4beta7 (α4β7) and alphaEbeta7(αEβ7)) are expressed primarily on monocytes, lymphocytes, eosinophils,basophils, and macrophages but not on neutrophils. Elices, M. J. et al.,Cell, 1990, 60:577-584. The primary ligands for α4β7 integrin are theendothelial surface proteins mucosal addressin cell adhesion molecule(MAdCAM) and vascular cell adhesion molecule (VCAM-1) (Makarem, R. etal., J. Biol. Chem., 1994, 269:4005-4011). The binding of the α4β7 toMAdCAM and/or VCAM expressed on high endothelial venules (HEVs) at sitesof inflammation results in firm adhesion of the leukocyte to theendothelium followed by extravasation into the inflamed tissue(Chuluyan, H. E. et al., Springer Semin. Immunopathol., 1995,16:391-404). A primary ligand for αEβ7 integrin is the intra-epitheliallymphocyte (IEL) surface protein, E-cadherein, which facilitatesadherence of the αEβ7-bearing cell to epithelial lymphocytes. Monoclonalantibodies directed against α4β7, MAdCAM or VCAM have been shown to beeffective modulators in animal models of chronic inflammatory diseasessuch as asthma (Laberge, S. et al., Am. J. Respir. Crit. Care Med.,1995, 151:822-829.), rheumatoid arthritis (R A; Barbadillo, C. et al.,Springer Semin. Immunopathol., 1995, 16:375-379), colitis (Viney et al,J. Immunol., 1996, 157: 2488-2497) and inflammatory bowel diseases (IBD;Podalski, D. K., N. Eng. J. Med., 1991, 325:928-937; Powrie, F. et al.,Ther. Immunol., 1995, 2:115-123). Monoclonal antibodies directed againstbeta7 subunit have been shown to bind the integrin subunit (Tidswell, M.et al. (1997) J. Immunol. 159:1497-1505) but as non-human ornon-humanized antibodies, they lack clinical usefulness.

A need exists for highly specific compounds, such as humanizedantibodies or binding fragments thereof which inhibit the interactionbetween the alpha4beta7 integrin and its ligands MAdCAM and/or VCAM aswell as the interaction between the alphaEbeta7 integrin and its ligandE-cadherin. These compounds are useful for treatment of chronicinflammatory diseases such as asthma, Crohn's disease, ulcerativecolitis, diabetes, complications of organ transplantation, andallograft-related disorders.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

DISCLOSURE OF THE INVENTION

The invention is in part based on the identification of a variety ofantagonists of biological pathways involving beta7-containing integrins,which are generally biological/cellular processes that presents as animportant and advantageous therapeutic target. Such biological pathwaysinclude, without limitation, inflammation, particularly chronicinflammation disorders such as asthma, allergy, IBD, diabetes,transplantation and grafts versus host disorders. The invention providescompositions and methods based on interfering with beta7integrin-mediated cellular adhesion and/or recruitment, including butnot limited to inferfering with MAdCAM and VCAM-1 binding to theextracellular portion of alpha4beta7 integrin and E-cadherin interactionwith the alphaEbeta7 integrin intereaction. Antagonists of theinvention, as described herein, provide important therapeutic anddiagnostic agents for use in targeting pathological conditionsassociated with abnormal or unwanted signaling via a beta7 integrin.Accordingly, the invention provides methods, compositions, kits andarticles of manufacture related to modulating beta7 integrin-mediatedpathways, including modulation of MAdCAM-alpha4beta7 binding andleukocyte recruitment to gastrointestinal epithelium, binding andallergy, asthma, IBD (such as Crohn's disease and ulcerative colitis),diabetes, inflammation associated with transplantation, graft versushost disorder and/or allograft disorders and otherbiological/physiological activities mediated by beta7 integrin.

In one aspect, the invention provides anti-beta7 therapeutic agentssuitable for therapeutic use and capable of effecting varying degrees ofdisruption of a beta7 integrin-mediated pathway. For example, in oneembodiment, the invention provides a humanized anti-beta7 antibodywherein the antibody as a Fab fragment has substantially the samebinding affinity to human beta7 as a murine Fab fragment comprising,consisting or consisting essentially of a light chain and heavy chainvariable domain sequence as depicted in FIGS. 1A and 1B or FIGS. 9A and9B. In another embodiment, the invention provides a humanized anti-beta7antibody wherein the antibody as a Fab fragment has a binding affinityto human beta7 that is lower, for example at least 3, at least 5, atleast 7 or at least 10-fold lower, than that of a murine or rat Fabfragment comprising, consisting or consisting essentially of a lightchain and heavy chain variable domain sequence as depicted in FIGS. 1Aand 1B or the variable domain sequences depicted in FIGS. 9A and 9B.Alternatively, a humanized anti-beta7 antibody, or beta7 bindingfragment thereof, of the invention exhibits monovalent affinity to humanbeta7, which affinity is substantially the same as or greater thanmonovalent affinity to human beta7 of an antibody comprising light chainand heavy chain variable sequences as depicted in FIG. 1A (SEQ ID NO:10)and/or FIG. 1B (SEQ ID NO:11), or FIG. 9A (SEQ ID NO:12) and/or FIG. 9B(SEQ ID NO:13). The antibody or binding fragment thereof having greataffinity to human beta7 exhibits an affinity which is at least 2-fold,at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold,at least 500-fold, at least 1000-fold, at least 5000-fold, or at least10,000-fold greater than an antibody comprising the light chain andheavy chain sequences depicted in FIG. 1A (SEQ ID NO:10) and/or FIG. 1B(SEQ ID NO:11), or FIG. 9A (SEQ ID NO:12) and/or FIG. 9B (SEQ ID NO:13).

In another embodiment, the invention provides an anti-beta7 humanizedantibody wherein the antibody as a Fab fragment has a binding affinityto human beta7 that is greater, for example at least 3, at least 5, atleast 7, at least 9, at least 10, at least 15, at least 20, or at least100-fold greater than that of a rodent (such as rat or murine) Fabfragment comprising, consisting or consisting essentially of a lightchain and heavy chain variable domain sequence as depicted in FIG. 1Aand FIG. 1B, respectively. In one embodiment, said rodent Fab fragmenthas the binding affinity of a Fab fragment comprising variable domainsequences of a rat antibody designated FIB504.64 produced by hybridomacell line deposited under American Type Culture Collection AccessionNumber ATCC HB-293. In a further embodiment, a humanized Fab fragment ofthe invention has the binding affinity of a Fab fragment comprisingvariable domain sequences of an antibody produced by anyone of thehumanized anti-beta7 antibodies of the invention. As is well-establishedin the art, binding affinity of a ligand to its receptor can bedetermined using any of a variety of assays, and expressed in terms of avariety of quantitative values. Accordingly, in one embodiment, thebinding affinity is expressed as Kd values and reflects intrinsicbinding affinity (e.g., with minimized avidity effects). Generally andpreferably, binding affinity is measured in vitro, whether in acell-free or cell-associated setting. As described in greater detailherein, fold difference in binding affinity can be quantified in termsof the ratio of the binding affinity value of a humanized antibody inFab form and the binding affinity value of a reference/comparator Fabantibody (e.g., a murine antibody having donor hypervariable regionsequences), wherein the binding affinity values are determined undersimilar assay conditions. Thus, in one embodiment, the fold differencein binding affinity is determined as the ratio of the Kd values of thehumanized antibody in Fab form and said reference/comparator Fabantibody. Any of a number of assays known in the art, including thosedescribed herein, can be used to obtain binding affinity measurements,including, for example, Biacore® (Biacore International Ab, Uppsala,Sweden) and ELISA.

In its various aspects and embodiments, the beta7 antagonist antibody ofthe invention is directed to the following set of potential claims forthis application: Antibody comprising an anti-beta7 antibody or beta7binding fragment thereof comprising:

(a) at least one, two, three, four, or five or hypervariable region(HVR) sequences selected from the group consisting of:

-   -   HVR-L1 comprising sequence A1-A11, wherein A1-A11 is RASESVDTYLH        (SEQ ID NO:1)    -   (ii) HVR-L2 comprising sequence B1-B8, wherein B1-B8 is KYASQSIS        (SEQ ID NO:2)    -   (iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is        QQGNSLPNT (SEQ ID NO:3)    -   (iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is        GFFITNNYWG (SEQ ID NO:4)    -   (v) HVR-H2 comprising sequence E1-E17, wherein E1-E17 is        GYISYSGSTSYNPSLKS (SEQ ID NO:5); and    -   (vi) HVR-H3 comprising sequence F2-F11, wherein F2-F11 is        MTGSSGYFDF (SEQ ID NO:6).

In an embodiment of the polypeptide or antibody of claim 1, thepolypeptide or antibody comprises at least one variant HVR, wherein thevariant HVR sequence comprises modification of at least one residue ofat least one of the sequences depicted in SEQ ID NOs:1, 2, 3, 4, 5, 6,7, 8, and 9. In another embodiment of claim 1 or claim 2, the inventioncomprises an an anti-beta7 antibody or beta7 binding fragment thereofcomprising one, two, three, four, five or six hypervariable regions(HVRs) selected from the group consisting of HVR-L1, HVR-L2, HVR-L3,HVR-H1, HVR-H2, and HVR-H3, wherein:

-   -   (i) HVR-L1 comprises amino acid sequence RASESVDTYLH (SEQ ID        NO:1); RASESVDSLLH (SEQ ID NO:7), RASESVDTLLH (SEQ ID NO:8), or        RASESVDDLLH (SEQ ID NO:9);    -   (ii) HVR-L2 comprises amino acid sequence KYASQSIS (SEQ ID        NO:2), RYASQSIS (SEQ ID NO:67, or XYASQSIS (SEQ ID NO:68, where        X represents any amino acid),    -   (iii) HVR-L3 comprises QQGNSLPNT (SEQ ID NO:3),    -   (iv) HVR-H1 comprises amino acid sequence GFFITNNYWG (SEQ ID        NO:4),    -   (v) HVR-H2 comprises amino acid sequence GYISYSGSTSYNPSLKS (SEQ        ID NO:5), and    -   (vi) HVR-H3 comprises amino acid sequence MTGSSGYFDF (SEQ ID        NO:6) or RTGSSGYFDF (SEQ ID NO:66) for relative positions        F2-F11; or comprises amino acid sequence F1-F11, wherein F1-F11        is AMTGSSGYFDF (SEQ ID NO:63), ARTGSSGYFDF (SEQ ID NO:64), or        AQTGSSGYFDF (SEQ ID NO:65).

In still another embodiment of claim 1 or any of the embodiments, theinvention comprises an an anti-beta7 antibody or beta7 binding fragmentthereof comprising one, two, three, four, five or six hypervariableregions (HVRs) selected from the group consisting of HVR-L1, HVR-L2,HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein:

-   -   (i) HVR-L1 comprises amino acid sequence A1-A11, wherein A1-A11        is RASESVDTYLH (SEQ ID NO:1); RASESVDSLLH (SEQ ID NO:7),        RASESVDTLLH (SEQ ID NO:8), or RASESVDDLLH (SEQ ID NO:9) or a        variant of SEQ ID NOs:1, 7, 8 or 9 wherein amino acid A2 is        selected from the group consisting of A, G, S, T, and V and/or        amino acid A3 is selected from the group consisting of S, G, I,        K, N, P, Q, R, and T, and/or A4 is selected from the group        consisting of E, V, Q, A, D, G, H, I, K, L, N, and R, and/or        amino acid A5 is selected from the group consisting of S, Y, A,        D, G, H, I, K, N, P, R, T, and V, and/or amino acid A6 is        selected from the group consisting of V, R, I, A, G, K, L, M,        and Q, and/or amino acid A7 is selected from the group        consisting of D, V, S, A, E, G, H, I, K, L, N, P, S, and T,        and/or amino acid A8 is selected from the group consisting of D,        G, N, E, T, P and S, and/or amino acid A9 is selected from the        group consisting of L, Y, I and M, and/or amino acid A10 is        selected from the group consisting of L, A, I, M, and V and/or        amino acid All is selected from the group consisting of H, Y, F,        and S;    -   (ii) HVR-L2 comprises amino acid sequence B1-B8, wherein B1-B8        is KYASQSIS (SEQ ID NO:2), RYASQSIS (SEQ ID NO:67, or XYASQSIS        (SEQ ID NO:68, where X represents any amino acid) or a variant        of SEQ ID NOs:2, 67 or 68 wherein amino acid B1 is selected from        the group consisting of K, R, N, V, A, F, Q, H, P, I, L, Y and X        (where X represents any amino acid), and/or amino acid B4 is        selected from the group consisting of S and D, and/or amino acid        B5 is selected from the group consisting of Q and S, and/or        amino acid B6 is selected from the group consisting of S, D, L,        and R, and/or amino acid B7 is selected from the group        consisting of I, V, E, and K;    -   (iii) HVR-L3 comprises amino acid sequence C1-C9, wherein C1-C9        is QQGNSLPNT (SEQ ID NO:3) or a variant of SEQ ID NO:3 wherein        amino acid C8 is selected from the group consisting of N, V, W,        Y, R, S, T, A, F, H, I, L, M, and Y;    -   (iv) HVR-H1 comprises amino acid sequence D1-D10 wherein D1-D10        is GFFITNNYWG (SEQ ID NO:4),    -   (v) HVR-H2 comprises amino acid sequence E1-E17 wherein E1-E17        is GYISYSGSTSYNPSLKS (SEQ ID NO:5), or a variant of SEQ ID NO:5        wherein amino acid E2 is selected from the group consisting of        Y, F, V, and D, and/or amino acid E6 is selected from the group        consisting of S and G, and/or amino acid E10 is selected from        the group consisting of S and Y, and/or amino acid E12 is        selected from the group consisting of N, T, A, and D, and/or        amino acid 13 is selected from the group consisting of P, H, D,        and A, and/or amino acid E15 is selected from the group        consisting of L and V, and/or amino acid E17 is selected from        the group consisting of S and G, and    -   (vi) HVR-H3 comprises amino acid sequence F2-F11 wherein F2-F11        is MTGSSGYFDF (SEQ ID NO:6) or RTGSSGYFDF (SEQ ID NO:66); or        comprises amino acid sequence F1-F11, wherein F1-F11 is        AMTGSSGYFDF (SEQ ID NO:63), ARTGSSGYFDF (SEQ ID NO:64), or        AQTGSSGYFDF (SEQ ID NO:65), or a variant of SEQ ID NOs:6, 63,        64, 65, or 66 wherein amino acid F2 is R, M, A, E, G, Q, S,        and/or amino acid F11 is selected from the group consisting of F        and Y.

In one embodiment of claim 1 or any of the antibodies of the invention,the amino acid at heavy chain framework position 71 (according to theKabat numbering system) is selected from the group consisting of R, A,and T, and/or the amino acid at heavy chain framework position 73 (Kabatnumbering system) is selected from the group consisting of N and T,and/or the amino acid at heavy chain framework position 78 (Kabatnumbering system) is selected from the group consisting of F, A, and L.

In one embodiment of claim 1 or any of the antibodies of the invention,HVR-L1 of an antibody of the invention comprises the sequence of SEQ IDNO:1. In one embodiment, HVR-L2 of an antibody of the inventioncomprises the sequence of SEQ ID NO:2. In one embodiment, HVR-L3 of anantibody of the invention comprises the sequence of SEQ ID NO:3. In oneembodiment, HVR-H1 of an antibody of the invention comprises thesequence of SEQ ID NO:4. In one embodiment, HVR-H2 of an antibody of theinvention comprises the sequence of SEQ ID NO:5. In one embodiment,HVR-H3 of an antibody of the invention comprises the sequence of SEQ IDNOs:6 or 66 for relative positions F2-F11 or SEQ ID NOs:63, 64, or 65for relative positions F1-F11. In one embodiment, HVR-L1 comprisesRASESVDSLLH (SEQ ID NO: 7). In one embodiment, HVR-L1 comprisesRASESVDTLLH (SEQ ID NO: 8). In one embodiment, HVR-L1 comprisesRASESVDDLLH (SEQ ID NO:9). In one embodiment, an antibody of theinvention comprising these sequences (in combinations as describedherein) is humanized or human.

In one aspect, the invention provides an antibody comprising one, two,three, four, five or six HVRs, wherein each HVR comprises, consists orconsists essentially of a sequence selected from the group consisting ofSEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8 and 9, and wherein SEQ ID NO:1, 7, 8or 9 corresponds to an HVR-L1, SEQ ID NO:2 corresponds to an HVR-L2, SEQID NO:3 corresponds to an HVR-L3, SEQ ID NO:4 corresponds to an HVR-H1,SEQ ID NO:5 corresponds to an HVR-H2, and SEQ ID NOs:6 corresponds to anHVR-H3. In one embodiment, an antibody of the invention comprisesHVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, inorder, comprises SEQ ID NO:1, 2, 3, 4, 5 and 6. In one embodiment, anantibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1,HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID NO:7, 2, 3,4, 5 and 6. In one embodiment, an antibody of the invention comprisesHVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, inorder, comprises SEQ ID NO:8, 2, 3, 4, 5 and 6. In one embodiment, anantibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1,HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID NO:9, 2, 3,4, 5 and 6. In one embodiment, an antibody of the invention comprisesHVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, inorder, comprises SEQ ID NO:9, 2, 3, 4, 5 and 66, or SEQ ID NO:9, 2, 3,4, 5, 63 or SEQ ID NO:9, 2, 3, 4, 5, 64 or SEQ ID NO:9, 2, 3, 4, 5, and65 or SEQ ID NO:9, 67, 3, 4, 5, 64 or SEQ ID NO:9, 68, 3, 4, 5, 64.

Variant HVRs in an antibody of the invention can have modifications ofone or more residues within the HVR and the HVRs and/or frameworkregions may be humanized. Embodiments of the invention in which there isan HVR and/or framework modification include, without limitation, thefollowing potential claims for this application:

-   2. The antibody of claim 1 or any of its embodiments, wherein A8 in    a variant HVR-L1 is S, D or T and A9 is L.-   3. The antibody of claim 1 or any of its embodiments, wherein the    antibody is humanized.-   4. The antibody of claim 1 or any of its embodiments, wherein at    least a portion of the framework sequence is a human consensus    framework sequence.-   5. The antibody of claim 1 or any of its embodiments, wherein said    modification is substitution, insertion or deletion.-   6. The antibody of claim 1 or any of its embodiments, wherein a    HVR-L1 variant comprises 1-10 (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)    substitutions in any combination of the following positions: A2 (G,    S, T, or V); A3 (G, I, K, N, P, Q, R, or T), A4 (A, D, G, H, I, K,    L, N, Q, R, or V), A5 (A, D, G, H, I, K, N, P, R, T, V, or Y), A6    (A, G, I, K, L, M, Q, or R), A7 (A, E, G, H, I, K, L, N, P, S, T, or    V), A8 (S, D, E, G, P, or N) and A9 (L, I, or M), A10 (A, I, M, or    V), and A11 (F, S, or Y).-   7. The antibody of claim 1 or any of its embodiments, wherein a    HVR-L2 variant comprises 1-4 (1, 2, 3, or 4) substitutions in any    combination of the following positions: B1 (N), B5 (S), B6 (R or L),    and B7 (T, E, K, or V).-   8. The antibody of claim 1 or any of its embodiments, wherein a    HVR-L3 variant comprises at least one substitution at position C8    (W, Y, R, S, A, F, H, I, L, M, N, T, or V).-   9. The antibody of claim 1 or any of its embodiments, wherein a    HVR-H2 variant comprises 1-7 (1, 2, 3, 4, 5, 6, or 7) substitutions    in any combination of the following positions: E2 (V, D, or F), E6    (G), E10 (Y), E12 (A, D, or T), E13 (D, A, or H), E15 (V), E17 (G).-   10. The antibody of claim 1 or any of its embodiments, wherein a    HVR-H3 variant comprises at 1 or 2 substitutions in any combination    of the following positions: F2 (A, E, G, Q, R, or S), and F11 (Y).-   11. The antibody of claim 1 or any of its embodiments, comprising an    HVR-L1 having the sequence of SEQ ID NO:7.-   12. The antibody of claim 1 or any of its embodiments, comprising an    HVR-L1 having the sequence of SEQ ID NO:8.-   13. The antibody of claim 1 or any of its embodiments, comprising an    HVR-L1 having the sequence of SEQ ID NO:9.-   14. The antibody of any one of claims 11-13 comprising a heavy chain    human subgroup III heavy chain consensus framework sequence    comprising a substitution at position 71, 73 and/or 78.-   15. The antibody of claim 14, wherein the substitution is R71A, N73T    and/or N78A.-   16. The antibody of claim 1 or any of its embodiments, comprising an    HVR-L3 having the sequence of SEQ ID NO:3.-   17. The antibody of claim 1 or any of its embodiments, wherein A8 in    a variant HVR-L1 is S.-   18. The antibody of claim 1 or any of its embodiments, wherein A8 in    a variant HVR-L1 is D.-   19. The antibody of claim 1 or any of its embodiments, wherein A9 in    a variant HVR-L1 is L.-   20. The antibody of claim 1 or any of its embodiments, wherein a    framework sequence between sequence E1-E17 and F1-F11 is    HFR3-1-HFR3-31 and wherein HFR3-6 is A or R, HFR3-8 is N or T, and    HFR3-13 is L or A or F.-   21. A humanized anti-beta7 antibody wherein monovalent affinity of    the antibody to human beta7 is substantially the same as monovalent    affinity of a rat antibody comprising a light chain and heavy chain    variable sequence as depicted in FIG. 9.-   22. A humanized anti-beta7 antibody wherein monovalent affinity of    the antibody to human beta7 is at least 3-fold greater than    monovalent affinity of a rat antibody comprising a light chain and    heavy chain variable sequence as depicted in FIG. 9.-   23. The humanized antibody of claim 21 or 22 wherein the rat    antibody is produced by hybridoma cell line deposited under American    Type Culture Collection Accession Number ATCC with designation    HB-293.-   24. The antibody of any of claims 21-23 wherein the binding affinity    is expressed as a Kd value.-   25. The antibody of any of claim 21-24 wherein the binding affinity    is measured by Biacore™ or radioimmunoassay.-   26. The antibody of claim 1 comprising human κ subgroup l light    chain consensus framework sequence.-   27. The antibody of claim 1 comprising heavy chain human subgroup    III heavy chain consensus framework sequence.-   28. The antibody of claim 27 wherein the framework sequence    comprises a substitution at position 71, 73 and/or 78.-   29. The antibody of claim 28 wherein said substitution is R71A, N73T    and/or N78A or wherein the substituted amino acid at position 71 is    R or A, and/or the amino acid substitution at position 78 is N or T,    and/or the amino acid substitution at position 78 is L or A or F.-   30. The antibody of claim 28 wherein said substitution is L78F or    A78F or A78L or L78A.-   31. A method of inhibiting the interaction of a human beta7 integrin    subunit with a second integrin subunit and/or a ligand by contacting    the antibody of any one of claims 1-30 with the second integrin    subunit and/or the ligand.-   32. The method of claim 31, wherein the second integrin subunit is    alpha4 integrin subunit, and wherein the ligand is MAdCAM, VCAM or    fibronectin.-   33. The method of claim 32, wherein the alpha4 integrin subunit is    human.-   34. The method of claim 33, wherein the ligand is human.-   35. The method of claim 32, wherein the second integrin subunit is    alphaE integrin subunit, and wherein the ligand is E-cadherein.-   36. The method of claim 35, wherein the alphaE integrin subunit is    human.-   37. The method of claim 36, wherein the ligand is human.-   38. The method of claim 31, wherein the inhibiting reduces or    alleviates symptoms of a disorder selected from inflammation,    asthma, inflammatory bowel disease, Crohn's disease, ulcerative    colitis, diabetes, inflammation resulting of organ transplantation,    graft versus host disorder, and inflammation associated with    allograft disorders.

Further embodiments of the invention include without limitation thefollowing:

In one embodiment, a HVR-L1 is SEQ ID NO:1, 7, 8, or 9 or a HVR-L1variant of SEQ ID NO:1, 7, 8, or 9 which comprises 1-10 (1, 2, 3, 4, 5,6, 7, 8, 9, or 10) substitutions at relative positions A1-A11, in anycombination of the following positions: A2 (A, G, S, T, or V); A3 (S, G,I, K, N, P, Q, R, or T), A4 (E, A, D, G, H, I, K, L, N, Q, R, or V), A5(S,A, D, G, H, I, K, N, P, R, T, V, or Y), A6 (V, A, G, I, K, L, M, Q,or R), A7 (D, A, E, G, H, I, K, L, N, P, S, T, or V), A8 (T, S, D, E, G,P, or N) and A9 (Y, L, I, or M), A10 (L, A, I, M, or V), and A11 (H, F,S, or Y). In one embodiment, a HVR-L2 is SEQ ID NO:2, 67, or 68 or aHVR-L2 variant of SEQ ID NO:2, 67, or 68 which HVR-L2 variant comprises1-4 (1, 2, 3, 4, 4 or 5) substitutions at relative positions B1-B8, inany combination of the following positions: B1 (K, R, N, V, A, F, Q, H,P, I, L, Y or X (where X represents any amino acid), B4 (S), B5 (Q orS), B6 (S, R or L), and B7 (I, T, E, K, or V). In one embodiment, aHVR-L3 is SEQ ID NO:3 or a HVR-L3 variant of SEQ ID NO:3 which comprisesat least one substitution at relative positions C1-C8, such as atposition C8 (W, Y, R, S, A, F, H, I, L, M, N, T, or V). In oneembodiment, a HVR-H1 is SEQ ID NO:4. In one embodiment, a HVR-H2 is SEQID NO:5 or a HVR-H2 variant of SEQ ID NO:5 which HVR-H2 variantcomprises 1-7 (1, 2, 3, 4, 5, 6, or 7) substitutions at relativepositions E1-E17 in any combination of the following positions: E2 (Y,V, D, or F), E6 (S or G), E10 (S or Y), E12 (N, A, D, or T), E13 (P, D,A, or H), E15 (L or V), E17 (S or G). In one embodiment, a HVR-H3 is SEQID NOs:6, 63, 64, 65, or 66 or a HVR-H3 variant of SEQ ID NOs:6, 63, 64,65, or 66 which comprises at relative positions F1-F11 for SEQ IDNOs:63, 64, and 65 or at relative positions F2-F11 for SEQ ID NOs:6 and66, 1 or 2 substitutions in any combination of the following positions:F2 (M, A, E, G, Q, R, or S), and F11 (F or Y). Letter(s) in parenthesisfollowing each position indicates an illustrative substitution (i.e.,replacement) amino acid for a consensus or other amino acid as would beevident to one skilled in the art, suitability of other amino acids assubstitution amino acids in the context described herein can beroutinely assessed using techniques known in the art and/or describedherein.

In one embodiment, a HVR-L1 comprises the sequence of SEQ ID NO:1. Inone embodiment, A8 in a variant HVR-L1 is D. In one embodiment, A8 in avariant HVR-L1 is S. In one embodiment, A9 in a variant HVR-L1 is L. Inone embodiment, A8 in a variant HVR-L1 is D and A9 in a variant HVR-L1is L. In one embodiment, A8 in a variant HVR-L1 is S and A9 in a variantHVR-L1 is L. In some embodiments of the invention comprises thesevariations in the HVR-L1, the HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3comprises or consists of or consists essentially of, in order, SEQ IDNO:2, 3, 4, 5, and 6. In some embodiments, HVR-H3 comprises or consistsor or consists essentially of SEQ ID NO:6 or 66 (for relative positionsF2-F11) or SEQ ID NO:63 or 64 or 65 (for relative positions F1-F11).

In one embodiment, A8 in a variant HVR-L1 is I and the and A9 in avariant HVR-L1 is L, which variant further comprises the HVR-L2, HVR-L3,HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting of, orconsist essentially of, in order, SEQ ID NO:2, 3, 4, 5, and 6.

In one embodiment, A8, A9, and A10 in a variant HVR-L1 are D, L, and V,respectively, which variant further comprises the HVR-L2, HVR-L3,HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting of, orconsist essentially of, in order, SEQ ID NO:2, 3, 4, 5, and 6.

In one embodiment, A8 and A9 in a variant HVR-L1 are N and L,respectively, which variant further comprises the HVR-L2, HVR-L3,HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting of, orconsist essentially of, in order, SEQ ID NO:2, 3, 4, 5, and 6.

In one embodiment, A8 and A9 in a variant HVR-L1 are P and L,respectively, and B6 and B7 in a variant HVR-L2 are R and T,respectively, which variant further comprises the HVR-L2, HVR-L3,HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting of, orconsist essentially of, in order, SEQ ID NO:3, 4, 5, and 6.

In one embodiment, A2, A4, A8, A9, and A10 in a variant HVR-L1 are S, D,S, L, and V, respectively, which variant further comprises the HVR-L2,HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting of,or consist essentially of, in order, SEQ ID NO:2, 3, 4, 5, and 6.

In one embodiment, A5 and A9 in a variant HVR-L1 are D and T,respectively, which variant further comprises the HVR-L2, HVR-L3,HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting of, orconsist essentially of, in order, SEQ ID NO:2, 3, 4, 5, and 6.

In one embodiment, A5 and A9 in a variant HVR-L1 are N and L,respectively, which variant further comprises the HVR-L2, HVR-L3,HVR-H1, HVR-H2, and HVR-H3, each HVR comprising, consisting of, orconsist essentially of, in order, SEQ ID NO:2, 3, 4, 5, and 6.

In one embodiment, A9 in a variant HVR-L1 is L, which variant furthercomprises the HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, each HVRcomprising, consisting of, or consist essentially of, in order, SEQ IDNO:2, 3, 4, 5, and 6.

In one embodiment, the antibody or anti-beta7 binding polypeptide of theinvention comprises an HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, andHVR-H3, each HVR comprising, consistingof, or consisting essentially of,in order, SEQ ID NO:9, 2, 3, 4, 5, and 64. In another embodiment, eachHVR comprises, consists of, or consists essentially of, in order, SEQ IDNO:9, 67, 3, 4, 5, and 64. In another embodiment, each HVR comprises,consists of, or consists essentially of, in order, SEQ ID NO:9, 68, 3,4, 5, and 64. In another embodiment, each HVR comprises, consists of, orconsists essentially of, in order, SEQ ID NO:9, 2 or 67 or 68, 3, 4, 5,and 66.

In some embodiments, said variant HVR-L1 antibody variants furthercomprises HVR-L2, HVR-L3, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, whereineach comprises, in order, the sequence depicted in SEQ ID NOs:2 3, 4, 5,and 6. Where the antibody variant comprises HVR-L1 A8(P) and A9(L) andHVR-L2 B6(R) and B7(T), in some embodiments said HVR-L1, HVR-L2 variantfurther comprises HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein eachcomprises, in order, the sequence depicted in SEQ ID NOs:3, 4, 5, and 6.

In some embodiments, these antibodies further comprise a human subgroupIII heavy chain framework consensus sequence. In one embodiment of theseantibodies, the framework consensus sequence comprises substitution atposition 71, 73 and/or 78. In some embodiments of these antibodies,position 71 is A, 73 is T and/or 78 is A. In one embodiment of theseantibodies, these antibodies further comprise a human κI light chainframework consensus sequence.

In one embodiment, an antibody of the invention comprises a HVR-L1comprising SEQ ID NO:1. In one embodiment, a variant antibody of theinvention comprises a variant HVR-L1 wherein A10 is V. In oneembodiment, said variant antibody further comprises HVR-L2, HVR-L3,HVR-H1, HVR-H2 and HVR-H3, wherein each comprises, in order, thesequence depicted in SEQ ID NOs:2, 3, 4, 5 and 6. In some embodiments,these antibodies further comprise a human subgroup III heavy chainframework consensus sequence. In one embodiment of these antibodies, theframework consensus sequence comprises substitution at position 71, 73and/or 78. In some embodiments of these antibodies, position 71 is A, 73is T and/or 78 is A. In one embodiment of these antibodies, theseantibodies further comprise a human κI light chain framework consensussequence.

In one embodiment, an antibody of the invention comprises a HVR-L3comprising SEQ ID NO:3. In one embodiment, a variant antibody of theinvention comprises a variant HVR-L3 wherein C8 is L. In one embodiment,said variant antibody further comprises HVR-L1, HVR-L2, HVR-H1, HVR-H2and HVR-H3, wherein each comprises, in order, the sequence depicted inSEQ ID NOs:1, 2, 4, 5 and 6. In one embodiment, an antibody of theinvention comprises a variant HVR-L3 wherein C8 is W. In one embodiment,said variant antibody further comprises HVR-L1, HVR-L2, HVR-H1, HVR-H2and HVR-H3, wherein each comprises, in order, the sequence depicted inSEQ ID NOs:1, 2, 4, 5 and 6. In some embodiment, HVR-L1 comprises SEQ IDNO:7, 8, or 9. In some embodiments, these antibodies further comprise ahuman subgroup III heavy chain framework consensus sequence. In oneembodiment of these antibodies, the framework consensus sequencecomprises substitution at position 71, 73 and/or 78. In some embodimentsof these antibodies, position 71 is A, 73 is T and/or 78 is A. In oneembodiment of these antibodies, these antibodies further comprise ahuman κI light chain framework consensus sequence.

In one embodiment, an antibody of the invention comprises a HVR-H3comprising SEQ ID NO:6. In one embodiment, a variant of said antibodycomprises a variant HVR-H3 wherein F1 is Q. In one embodiment, saidvariant antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1 andHVR-H2, wherein each comprises, in order, the sequence depicted in SEQID NOs:1, 2, 3, 4, and 5. In one embodiment, an antibody of theinvention comprises a variant HVR-H3 wherein F1 is R. In one embodiment,said variant antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1and HVR-H2, wherein each comprises, in order, the sequence depicted inSEQ ID NOs:1, 2, 3, 4, and 5. In one embodiment, HVR-L1 comprises SEQ IDNO:7, 8, or 9. In some embodiments, these antibodies further comprise ahuman subgroup III heavy chain framework consensus sequence. In oneembodiment of these antibodies, the framework consensus sequencecomprises substitution at position 71, 73 and/or 78. In some embodimentsof these antibodies, position 71 is A, 73 is T and/or 78 is A. In oneembodiment of these antibodies, these antibodies further comprise ahuman κI light chain framework consensus sequence.

In one embodiment, an antibody of the invention comprises a HVR-L1comprising SEQ ID NO:1. In one embodiment, the antibody comprises avariant HVR-L1 wherein A4 is Q. In one embodiment, said variant antibodyfurther comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, whereineach comprises, in order, the sequence depicted in SEQ ID NOs:2, 3, 4,5, and 6. In one embodiment, an antibody of the invention comprises avariant HVR-L1 wherein A6 is I. In one embodiment, said variant antibodyfurther comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, whereineach comprises, in order, the sequence depicted in SEQ ID NOs:2, 3, 4,5, and 6. In one embodiment, an antibody of the invention comprises avariant HVR-L1 wherein A7 is S. In one embodiment, said variant antibodyfurther comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, whereineach comprises, in order, the sequence depicted in SEQ ID NOs:2, 3, 4,5, and 6. In one embodiment, an antibody of the invention comprises avariant HVR-L1 wherein A8 is D or N. In one embodiment, said variantantibody further comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3,wherein each comprises, in order, the sequence depicted in SEQ ID NOs:2,3, 4, 5, and 6. In some embodiments, these antibodies further comprise ahuman subgroup III heavy chain framework consensus sequence. In oneembodiment of these antibodies, the framework consensus sequencecomprises substitution at position 71, 73 and/or 78. In some embodimentsof these antibodies, position 71 is A, 73 is T and/or 78 is A. In oneembodiment of these antibodies, these antibodies further comprise ahuman κI light chain framework consensus sequence.

In one embodiment, an antibody of the invention comprises a HVR-L2comprising SEQ ID NO:2. In one embodiment, an antibody of the inventioncomprises a variant HVR-L2 wherein B1 is N. In one embodiment, anantibody of the invention comprises a variant HVR-L2 wherein B5 is S. Inone embodiment, an antibody of the invention comprises a variant HVR-L2wherein B6 is L. In one embodiment, an antibody of the inventioncomprises a variant HVR-L2 wherein B7 is V. In one embodiment, anantibody of the invention comprises a variant HVR-L2 wherein B7 is E orK. In some embodiments, said variant antibody further comprises HVR-L1,HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each comprises, in order, thesequence depicted in SEQ ID NOs:1, 3, 4, 5, and 6. In some embodiments,HVR-L1 comprises SEQ ID NO:7, 8, or 9. In some embodiments, theseantibodies further comprise a human subgroup III heavy chain frameworkconsensus sequence. In one embodiment of these antibodies, the frameworkconsensus sequence comprises substitution at position 71, 73 and/or 78.In some embodiments of these antibodies, position 71 is A, 73 is Tand/or 78 is A. In one embodiment of these antibodies, these antibodiesfurther comprise a human κI light chain framework consensus sequence.

In one embodiment, an antibody of the invention comprises a HVR-L3comprising SEQ ID NO:3. In one embodiment, an antibody of the inventioncomprises a variant HVR-L3 wherein C8 is W, Y, R, or S. In someembodiments, said variant antibody further comprises HVR-L1, HVR-L2,HVR-H1, HVR-H2 and HVR-H3, wherein each comprises, in order, thesequence depicted in SEQ ID NOs:1, 2, 4, 5, and 6. In some embodiments,HVR-L1 comprises SEQ ID NO:7, 8, or 9. In some embodiments, theseantibodies further comprise a human subgroup III heavy chain frameworkconsensus sequence. In one embodiment of these antibodies, the frameworkconsensus sequence comprises substitution at position 71, 73 and/or 78.In some embodiments of these antibodies, position 71 is A, 73 is Tand/or 78 is A. In one embodiment of these antibodies, these antibodiesfurther comprise a human κI light chain framework consensus sequence.

In one embodiment, an antibody of the invention comprises a HVR-H2comprising SEQ ID NO:5. In one embodiment, an antibody of the inventioncomprises a variant HVR-H2 wherein E2 is F. In one embodiment, anantibody of the invention comprises a variant HVR-H2 wherein E2 is V orD. In one embodiment, an antibody of the invention comprises a variantHVR-H2 wherein E6 is G. In one embodiment, an antibody of the inventioncomprises a variant HVR-H2 wherein E10 is Y. In one embodiment, anantibody of the invention comprises a variant HVR-H2 wherein E12 is A,D, or T. In one embodiment, an antibody of the invention comprises avariant HVR-H2 wherein E13 is D, A, or N. In one embodiment, an antibodyof the invention comprises a variant HVR-H2 wherein E15 is V. In oneembodiment, an antibody of the invention comprises a variant HVR-H2wherein E17 is G. In some embodiments, said variant antibody furthercomprises HVR-L1, HVR-L2, HVR-L3, HVR-H1 and HVR-H3, wherein eachcomprises, in order, the sequence depicted in SEQ ID NOs:1, 2, 3, 4, and6. In some embodiments, HVR-L1 comprises SEQ ID NO:7, 8, or 9. In someembodiments, these antibodies further comprise a human subgroup IIIheavy chain framework consensus sequence. In one embodiment of theseantibodies, the framework consensus sequence comprises substitution atposition 71, 73 and/or 78. In some embodiments of these antibodies,position 71 is A, 73 is T and/or 78 is A. In one embodiment of theseantibodies, these antibodies further comprise a human κI light chainframework consensus sequence.

In one embodiment, an antibody of the invention comprises a HVR-H3comprising SEQ ID NO:6. In one embodiment, an antibody of the inventioncomprises a variant HVR-H3 wherein F11 is Y. In some embodiments, saidvariant antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1 andHVR-H3, wherein each comprises, in order, the sequence depicted in SEQID NOs:1, 2, 3, 4, and 6. In some embodiments, HVR-L1 comprises SEQ IDNO:7, 8, or 9. In some embodiments, these antibodies further comprise ahuman subgroup III heavy chain framework consensus sequence. In oneembodiment of these antibodies, the framework consensus sequencecomprises substitution at position 71, 73 and/or 78. In some embodimentsof these antibodies, position 71 is A, 73 is T and/or 78 is A. In oneembodiment of these antibodies, these antibodies further comprise ahuman κI light chain framework consensus sequence.

In some embodiments, these antibodies further comprise a human subgroupIII heavy chain framework consensus sequence. In one embodiment of theseantibodies, the framework consensus sequence comprises substitution atposition 71, 73 and/or 78. In some embodiments of these antibodies,position 71 is A, 73 is T and/or 78 is A. In one embodiment of theseantibodies, these antibodies further comprise a human κI light chainframework consensus sequence.

A therapeutic agent for use in a host subject preferably elicits littleto no immunogenic response against the agent in said subject. In oneembodiment, the invention provides such an agent. For example, in oneembodiment, the invention provides a humanized antibody that elicitsand/or is expected to elicit a human anti-rodent antibody response (suchas anti-mouse or anti-rat response) or a human anti-human response at asubstantially reduced level compared to an antibody comprising thesequence comprising SEQ ID NOs:10 and/or 11 (FIGS. 1A and 1B) or SEQ IDNOs: 12 and/or 13 (FIGS. 9A and 9B depicting rat anti-mouse Fib504 aminoacid sequences) in a host subject. In another example, the inventionprovides a humanized antibody that elicits and/or is expected to elicitno human anti-rodent (such as human anti-mouse (HAMA) or humananti-mouse) or human anti-human antibody response (HAHA).

A humanized antibody of the invention may comprise one or more humanand/or human consensus non-hypervariable region (e.g., framework)sequences in its heavy and/or light chain variable domain. In someembodiments, one or more additional modifications are present within thehuman and/or human consensus non-hypervariable region sequences. In oneembodiment, the heavy chain variable domain of an antibody of theinvention comprises a human consensus framework sequence, which in oneembodiment is the subgroup III consensus framework sequence. In oneembodiment, an antibody of the invention comprises a variant subgroupIII consensus framework sequence modified at at least one amino acidposition. For example, in one embodiment, a variant subgroup IIIconsensus framework sequence may comprise a substitution at one or moreof positions 71, 73, 78 and/or 94. In one embodiment, said substitutionis R71A, N73T, L78A, and/or R94M, in any combination thereof.

As is known in the art, and as described in greater detail hereinbelow,the amino acid position/boundary delineating a hypervariable region ofan antibody can vary, depending on the context and the variousdefinitions known in the art (as described below). Some positions withina variable domain may be viewed as hybrid hypervariable positions inthat these positions can be deemed to be within a hypervariable regionunder one set of criteria while being deemed to be outside ahypervariable region under a different set of criteria. One or more ofthese positions can also be found in extended hypervariable regions (asfurther defined below). The invention provides antibodies comprisingmodifications in these hybrid hypervariable positions. In oneembodiment, these hybrid hypervariable positions include one or more ofpositions 26-30, 33-35B, 47-49, 49, 57-65, 93, 94 and 102 in a heavychain variable domain. In one embodiment, these hybrid hypervariablepositions include one or more of positions 24-29, 35-36, 46-49, 49, 56and 97 in a light chain variable domain. In one embodiment, an antibodyof the invention comprises a variant human subgroup consensus frameworksequence modified at one or more hybrid hypervariable positions. In oneembodiment, an antibody of the invention comprises a heavy chainvariable domain comprising a variant human subgroup III consensusframework sequence modified at one or more of positions 28-35, 49, 50,52a, 53, 54, 58-61, 63, 65, 94 and 102. In one embodiment, the antibodycomprises a, T28F, F29I, S30T, S31N, Y32N, A33Y, M34W, and S35Gsubstitution. In one embodiment, the antibody comprises a S49Gsubstitution. In one embodiment, the antibody comprises a V50F or V50Dor V50Y substitution. In one embodiment, the antibody comprises a G53Ysubstitution. In one embodiment, the antibody comprises a G54Ssubstitution. In one embodiment, the antibody comprises a Y58Ssubstitution. In one embodiment, the antibody comprises a A60N or A60Dor A60T substitution. In one embodiment, the antibody comprises a D61Por D61A or D61H substitution. In one embodiment, the antibody comprisesa V63L substitution. In one embodiment, the antibody comprises a G65Ssubstitution. In one embodiment, the antibody comprises a R94Msubstitution. In one embodiment, the antibody comprises a R94A or R94Eor R94G or R94Q or R94S substitution. In one embodiment, the antibodycomprises a G95T substitution. In one embodiment, the antibody comprisesone or more of the substitutions at positions 28-35, 49, 50, 52a, 53,54, 58-61, 63, 65, 94 and 102 and further comprises one or more of thesubstitutions at positions R71A or N73T or L78A or L78F. In oneembodiment, the antibody comprises a Y102F substitution. It can be seenby reference to FIG. 1B that these substitutions are in the HVR-H1,HVR-H2, and/or HVR-H3 of the heavy chain.

In one embodiment, an antibody of the invention comprises a light chainvariable domain comprising a variant human subgroup I consensusframework sequence modified at one or more of positions 27, 29-31, 33,34, 49, 50, 53-55, 91 and 96. In one embodiment, the antibody comprisesa Q27E substitution. In one embodiment, the antibody comprises a I29Vsubstitution. In one embodiment, the antibody comprises a S30Dsubstitution. In one embodiment, the antibody comprises a N31T or N31Sor N31D substitution. In one embodiment, the antibody comprises a Y32L.In one embodiment, the antibody comprises a A34H substitution. In oneembodiment, the antibody comprises a Y49K substitution. In oneembodiment, the antibody comprises a A50Y substitution. In oneembodiment, the antibody comprises a S53Q substitution. In oneembodiment, the antibody comprises a L54S substitution. In oneembodiment, the antibody comprises a E55I or E55V substitution. In oneembodiment, the antibody comprises a Y91G substitution. In oneembodiment, the antibody comprises a W96N or W96L substitution. In oneembodiment, the antibody comprises a A25S substitution. In oneembodiment, the antibody comprises a A25 to G, S, T, or V substitution.In one embodiment, the antibody comprises a modification selected fromone or more of the following groups of substitutions. For example, inone embodiment, the antibody comprises a S26 to G, I, K, N, P, Q, or Tsubstitution. In one embodiment, the antibody comprises a Q27 to E, A,D,G, H, I, K, L, N, Q, R, or V substitution. In one embodiment, theantibody comprises a S28 to A, D, G, H, I, K, N, P, R, T, V, or Ysubstitution. In one embodiment, the antibody comprises a I29 to V, A,G, K, L, M, Q or R substitution. In one embodiment, the antibodycomprises a S30 to D, A, E, G, H, I, K, L, N, P, S, T or V substitution.In one embodiment, the antibody comprises a N31 to D, T, E, or Gsubstitution. In one embodiment, the antibody comprises a Y32 to L, I orM substitution. In one embodiment, the antibody comprises a L33 to A, I,M or V substitution. In one embodiment, the antibody comprises a A34 toH, F, Y or S substitution. In one embodiment, the antibody comprises aY49 to K or N substitution. In one embodiment, the antibody comprises aA50Y substitution. In one embodiment, the antibody comprises S53Qsubstitution. In one embodiment, the antibody comprises a L54Ssubstitution. In one embodiment, the antibody comprises a E55 to V, I orK substitution. In one embodiment, the antibody comprises a Y91Gsubstitution. In one embodiment, the antibody comprises a W96 to N, L,W, Y, R, S, A, F, H, I, M, N, R, S, T, V or Y substitution. It can beseen by reference to FIG. 1A that these substitutions are in the HVR-L1,HVR-L2, and/or HVR-L3 of the light chain.

An antibody of the invention can comprise any suitable human or humanconsensus light chain framework sequences, provided the antibodyexhibits the desired biological characteristics (e.g., a desired bindingaffinity). In one embodiment, an antibody of the invention comprises atleast a portion (or all) of the framework sequence of human κ lightchain. In one embodiment, an antibody of the invention comprises atleast a portion (or all) of human κ subgroup I framework consensussequence.

In one embodiment, an antibody of the invention comprises a heavy and/orlight chain variable domain comprising framework sequences depicted SEQID NOS:34-41 and in FIGS. 1, 7 and 8, provided positions 49 of the lightchain and 94 of the heavy chain are included in the extended HVRs, andprovided said position 49 is K and said position 94 is preferably butnot necessarily M and may be R.

Antagonists of the invention can be used to modulate one or more aspectsof beta7 associated effects, including but not limited to associationwith alpha4 integrin subunit, association with alphaE integrin subunit,binding of alpha4beta7 integrin to MAdCAM, VCAM-1 or fibronectin andbinding of alphaEbeta7 integrin to E-caderin. These effects can bemodulated by any biologically relevant mechanism, including disruptionof ligand binding to beta7 subunit or to the alpha4beta7 or alphaEbetadimeric integrin, and/or by disrupting association between the alpha andbeta integrin subunits such that formation of the dimeric integrin isinhibited. Accordingly, in one embodiment, the invention provides abeta7 antagonist antibody that inhibits binding of alpha4 to beta7. Inone embodiment, a beta7 antagonist antibody of the invention disruptsbinding of alpha4beta7 to MAdCAM. In one embodiment, a beta7 antagonistantibody of the invention disrupts binding of alpha4beta7 to VCAM-1. Inone embodiment, a beta7 antagonist antibody of the invention disruptsbinding of alpha4beta7 to fibronectin. In one embodiment, a beta7antagonist antibody of the invention disrupts binding of beta7 toalphaE. In one embodiment, a beta7 antagonist antibody of the inventiondisrupts binding alphaEbeta7 integrin to E-cadherin. Interference can bedirect or indirect. For example, a beta7 antagonist antibody may bind tobeta7 within a sequence of the alpha4beta7 or alphaEbeta7 dimerizationregion, and thereby inhibit interaction of the integrin subunits andformation of an integrin dimer. In a further example, a beta7 antagonistantibody may bind to a sequence within the ligand binding domain ofbeta7 subunit and thereby inhibit interaction of said bound domain withits binding partner (such as fibronectin, VCAM, and/or MAdCAM for thealpha4beta7 integrin; or E-cadherin for the alphaEbeta7 integrin). Inanother example, a beta7 antagonist antibody may bind to a sequence thatis not within the integrin subunit dimerization domain or a ligandbinding domain, but wherein said beta7 antagonist antibody bindingresults in disruption of the ability of the beta7 domain to interactwith its binding partner (such as an alpha4 or alphaE integrin subunitand/or a ligand such as fibronectin, VCAM, MAdCAM, or E-cadherein). Inone embodiment, an antagonist antibody of the invention binds to beta7(for example, the extracellular domain) such that beta7 dimerizationwith the alpha4 or alphaE subunit is disrupted. In one embodiment, anantagonist antibody of the invention binds to beta7 such that ability ofbeta7 and/or an alpha4beta7 and/or an alphaEbeta7 integrin to bind toits respective ligand or ligands is disrupted. For example, in oneembodiment, the invention provides an antagonist antibody which uponbinding to a beta7 molecule inhibits dimerization of said molecule. Inone embodiment, a beta7 antagonist antibody of the inventionspecifically binds a sequence in the ligand binding domain of beta7. Inone embodiment, a beta7 antagonist antibody of the inventionspecifically binds a sequence in the ligand binding domain of beta7 suchthat ligand binding (i.e., fibronectin, VCAM, and/or MAdCAM) to thealpha4beta7 integrin is disrupted. In one embodiment, a beta7 antagonistantibody of the invention specifically binds a sequence in the ligandbinding domain of beta7 such that ligand binding (i.e., E-cadherin) tothe alphaEbeta7 integrin is disrupted.

In one embodiment, an antagonist antibody of the invention disruptsbeta7 dimerization comprising heterodimerization (i.e., beta7dimerization with an alpha4 or alphaE integrin subunit molecule).

In one embodiment, an antagonist antibody of the invention binds to anepitope on the beta7 integrin subunit that maps to amino acids 176-237.In another embodiment, an antagonist antibody of the invention binds tothe same epitope on the beta7 integrin that is the substantially thesame epitope as Fib504.64 (ATCC HB-293). Determination of epitopebinding is by standard techniques including without limitationcompetition binding analysis.

In one aspect, the invention provides an antibody comprising acombination of one, two, three, four, five or all of the HVR sequencesdepicted in the table of amino acid substitutions in FIG. 13.

A therapeutic agent for use in a host subject preferably elicits littleto no immunogenic response against the agent in said subject. In oneembodiment, the invention provides such an agent. For example, in oneembodiment, the invention provides a humanized antibody that elicitsand/or is expected to elicit a human anti-rat or human anti-mouse orhuman anti-human antibody response at a substantially reduced levelcompared to an antibody comprising the sequence of SEQ ID NOS:10, 11, 12and/or SEQ ID NO:13 (rat anti-mouse Fib504 (ATCC HB-293), FIGS. 1 and 9)in a host subject. In another example, the invention provides ahumanized antibody that elicits and/or is expected to elicit no humananti-mouse, human anti-rat, or human anti-human antibody response.

A humanized antibody of the invention may comprise one or more humanand/or human consensus non-hypervariable region (e.g., framework)sequences in its heavy and/or light chain variable domain. In someembodiments, one or more additional modifications are present within thehuman and/or human consensus non-hypervariable region sequences. In oneembodiment, the heavy chain variable domain of an antibody of theinvention comprises a human consensus framework sequence, which in oneembodiment is the subgroup III consensus framework sequence. In oneembodiment, an antibody of the invention comprises a variant subgroupIII consensus framework sequence modified at at least one amino acidposition. For example, in one embodiment, a variant subgroup IIIconsensus framework sequence may comprise a substitution at one or moreof positions 71, 73, 78 and/or 94, although position 94 is part of anextended heavy chain hypervariable region-H3 of the present invention.In one embodiment, said substitution is R71A, N73T, N78A, and/or R94M,in any combination thereof.

An antibody of the invention can comprise any suitable human or humanconsensus light chain framework sequences, provided the antibodyexhibits the desired biological characteristics (e.g., a desired bindingaffinity). In one embodiment, an antibody of the invention comprises atleast a portion (or all) of the framework sequence of human κ lightchain. In one embodiment, an antibody of the invention comprises atleast a portion (or all) of human κ subgroup I framework consensussequence.

Antagonists of the invention can be used to modulate one or more aspectsof beta7 associated effects. For example, a beta7 antagonist antibodymay bind to beta7 within a sequence of the alpha4beta7 or alphaEbeta7dimerization region, and thereby inhibit interaction of the integrinsubunits and formation of an integrin dimer. In a further example, abeta7 antagonist antibody may bind to a sequence within the ligandbinding domain of beta7 subunit and thereby inhibit interaction of saidbound domain with its binding partner (such as fibronectin, VCAM, and/orMAdCAM for the alpha4beta7 integrin; or E-cadherin for the alphaEbeta7integrin). In another example, a beta7 antagonist antibody may bind to asequence that is not within the integrin subunit dimerization domain ora ligand binding domain, but wherein said beta7 antagonist antibodybinding results in disruption of the ability of the beta7 domain tointeract with its binding partner (such as an alpha4 or alphaE integrinsubunit and/or a ligand such as fibronectin, VCAM, MAdCAM, orE-cadherein). In one embodiment, an antagonist antibody of the inventionbinds to beta7 (for example, the extracellular domain) such that beta7dimerization with the alpha4 or alphaE subunit is disrupted. In oneembodiment, an antagonist antibody of the invention binds to beta7 suchthat ability of beta7 and/or an alpha4beta7 and/or an alphaEbeta7integrin to bind to its respective ligand or ligands is disrupted. Forexample, in one embodiment, the invention provides an antagonistantibody which upon binding to a beta7 molecule inhibits dimerization ofsaid molecule. In one embodiment, a beta7 antagonist antibody of theinvention specifically binds a sequence in the ligand binding domain ofbeta7. In one embodiment, a beta7 antagonist antibody of the inventionspecifically binds a sequence in the ligand binding domain of beta7 suchthat ligand binding (i.e., fibronectin, VCAM, and/or MAdCAM) to thealpha4beta7 integrin is disrupted. In one embodiment, a beta7 antagonistantibody of the invention specifically binds a sequence in the ligandbinding domain of beta7 such that ligand binding (i.e., E-cadherin) tothe alphaEbeta7 integrin is disrupted.

In one embodiment, an antagonist antibody of the invention disruptsbeta7 dimerization comprising heterodimerization (i.e., beta7dimerization with an alpha4 or alphaE integrin subunit molecule.

In some instances, it may be advantageous to have a beta7 antagonistantibody that does not interfere with binding of a ligand (such asfibronectin, VCAM, MAdCAM, or alphaE) to beta7 subunit as part of anintegrin or to an alpha4beta7 integrin or an alphaEbeta7 integrin as adimer. Accordingly, in one embodiment, the invention provides anantibody that does not bind a fibronectin, VCAM, MAdCAM, or E-cadherinbinding site on beta7 but, instead, inhibits interaction between beta7subunit and an alpha subunit (such as alpha4 or alphaE integrin subunit)such that a biologically active integrin is prevented from forming. Inone example, an antagonist antibody of the invention can be used inconjunction with one or more other antagonists, wherein the antagonistsare targeted at different processes and/or functions within the beta7integrin axis. Thus, in one embodiment, a beta7 antagonist antibody ofthe invention binds to an epitope on beta7 distinct from an epitopebound by another beta7 or an alpha/beta integrin antagonist (such as analpha4beta7 antibody, including monoclonal antibody or an antibody, suchas a humanized antibody or monoclonal antibody derived from and/orhaving the same or effectively the same binding characteristics orspecificity as an antibody derived from a murine antibody.

In one embodiment, the invention provides a beta7 antagonist antibodythat disrupts beta7-alpha4 or -alphaE multimerization into therespective integrin as well as ligand binding. For example, anantagonist antibody of the invention that inhibits beta7 dimerizationwith alpha4 or alphaE integrin subunit may further comprise an abilityto compete with ligand for binding to beta7 or the integrin dimer (e.g.,it may interfere with the binding of fibronectin, VCAM, and/or MAdCAM tobeta7 and/or alpha4beta7; or it may interfere with the binding ofE-cadherin to beta7 or alphaEbeta7.)

In one embodiment of a beta7 antagonist antibody of the invention,binding of the antagonist to beta7 inhibits ligand binding activatedcellular adhesion. In another embodiment of a beta7 antagonist antibodyof the invention, binding of the antagonist to beta7 in a cell inhibitsrecruitment of the cell to the cells and/or tissue in which thebeta7-containing integrin is expressed.

In one embodiment, a beta7 antagonist antibody of the inventionspecifically binds at least a portion of amino acids 176-250 (optionallyamino acids 176-237) of the beta7 extracellular domain (see Tidswell, M.et al. (1997) J. Immunol. 159:1497-1505) or variant thereof, and reducesor blocks binding of ligands MAdCAM, VCAM-1, fibronectin, and/orE-cadherin. In one embodiment, such blocking of ligand binding disrupts,reduces and/or prevents adhesion of a cell expressing the ligand to acell expressing the beta7-containing ligand. In one embodiment, anantagonist antibody of the invention specifically binds an amino acidsequence of beta7 comprising residues 176-237. In one embodiment, anantagonist antibody of the invention specifically binds a conformationalepitope formed by part or all of at least one of the sequences selectedfrom the group consisting of residues 176-237 of beta7. In oneembodiment, an antagonist antibody of the invention specifically bindsan amino acid sequence having at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, at least 98%, at least 99%sequence identity or similarity with the amino acid sequence of residues176-237 or residues176-250 of human beta7. In one embodiment, theantagonist anti-beta7 antibody of the invention binds the same epitopeas the anti-beta7 antibody Fib504 produced by hybridoma ATCC HB-293.

In one aspect, the invention provides compositions comprising one ormore antagonist antibodies of the invention and a carrier. In oneembodiment, the carrier is pharmaceutically acceptable.

In one aspect, the invention provides nucleic acids encoding a beta7antagonist antibody of the invention.

In one aspect, the invention provides vectors comprising a nucleic acidof the invention.

In one aspect, the invention provides host cells comprising a nucleicacid or a vector of the invention. A vector can be of any type, forexample a recombinant vector such as an expression vector. Any of avariety of host cells can be used. In one embodiment, a host cell is aprokaryotic cell, for example, E. coli. In one embodiment, a host cellis a eukaryotic cell, for example a mammalian cell such as ChineseHamster Ovary (CHO) cell.

In one aspect, the invention provides methods for making an antagonistof the invention. For example, the invention provides a method of makinga beta7 antagonist antibody (which, as defined herein includes fulllength and fragments thereof), said method comprising expressing in asuitable host cell a recombinant vector of the invention encoding saidantibody (or fragment thereof), and recovering said antibody.

In one aspect, the invention provides an article of manufacturecomprising a container; and a composition contained within thecontainer, wherein the composition comprises one or more beta7antagonist antibodies of the invention. In one embodiment, thecomposition comprises a nucleic acid of the invention. In oneembodiment, a composition comprising an antagonist antibody furthercomprises a carrier, which in some embodiments is pharmaceuticallyacceptable. In one embodiment, an article of manufacture of theinvention further comprises instructions for administering thecomposition (for example, the antagonist antibody) to a subject.

In one aspect, the invention provides a kit comprising a first containercomprising a composition comprising one or more beta7 antagonistantibodies of the invention; and a second container comprising a buffer.In one embodiment, the buffer is pharmaceutically acceptable. In oneembodiment, a composition comprising an antagonist antibody furthercomprises a carrier, which in some embodiments is pharmaceuticallyacceptable. In one embodiment, a kit further comprises instructions foradministering the composition (for example, the antagonist antibody) toa subject.

Beta7 integrins and their ligands are variously expressed in diseasestates. [The expression of MAdCAM-1 on gut endothelium is increased insites of mucosal inflammation in patients with inflammatory boweldisease (UC and CD) and colonic lamina propria of UC and CD patientsalso show increased CD3+ and a4b7+ cells compared to IBS controls (seeSouza H., et al., Gut 45:856 (1999)). MAdCAM-1 expression was observedto be associated with portal tract inflammation in liver diseases andmay be important in recruitment of alpha4beta7+ lymphocytes to the liverduring inflammation. (Hillan, K., et al., Liver. 19(6):509-18 (1999))MAdCAM-1 on hepatic vessels supports adhesion of a4b7+ lymphocytes frompatients with IBD and primary sclerosing cholangitis. The adhesion wasinhibited by anti-MAdCAM-1, anti-alpha4beta7, or anti-alpha4 antibodies.(Grant A J. et al., Hepatology. 33(5):1065-72 (2001)). MAdCAM-1, VCAM-1and E-cadherin are expressed on brain endothelial cells and/or onmicrovessels in the inflamed central nervous system. Beta7 integrinscontribute to demyelinating disease of the CNS (Kanwar et al., J.Neuroimmunology 103, 146 (2000)). Expression of alpha4beta7 wassignificantly higher in the LPL of CD than in controls and patients withUC (Oshitani, N. et al., International Journal of Molecule Medicine 12,715-719 (2003)). IELs from CD patients may be chronically stimulated andrecruited from the periphery (Meresse, B., et al., Human Immunology, 62,694-700 (2001)). In human liver disease, alphaEbeta7 T cells (CD4+ andCD8+) are preferentially accumulated in human livers where E-cadherin isexpressed on hepatocytes and bile duct epithelium (Shimizu, Y., et al.,Journal of Hepatology 39, 918-924 (2003)). In chronic pancreatitis,CD8+CD103+ T cells, analogous to intestinal intraepithelial lymphocytes,infiltrate the pancreas in chronic pancreatitis (Matthias, P., et al.,Am J Gastroenterol 93:2141-2147 (1998)). Upregulation of alphaEbeta7 isfound in systemic lupus erythematosus patients with specific epithelialinvolvement (Pang et al., Arthritis & Rheumatism 41:1456-1463 (1998)).In Sjogren's Syndrome, CD8+ alphaEbeta7+ T cells adhere and kill acinarepithelial cells by inducing apoptosis (Kroneld et al., Scand JRheumatol 27:215-218, 1998) Integrin alpha4beta7 and alphaEbeta7 play arole in T cell epidermotropism during skin inflammation and contributeto skin allograft rejection (Sun et al., Transplantation 74, 1202,2002). Teraki and Shiohara showed preferential expression of aEb7integrin on CD8+ T cells in psoriatic epidermis (Teraki and Shiohara,Br. J. Dermatology 147, 1118, 2002). Sputum T lymphocytes are activatedIELs (CD69+ CD103+) in asthma, COPD, and normal subjects (Leckie et.al., Thorax 58, 23, 2003). CD103+ (aEb7+) CTL accumulate with graftepithelium during clincial renal allograft rejection (Hadley et al.,Transplantation 72, 1548, 2001)] Thus, in one aspect, the inventionprovides use of a beta7 antagonist antibody of the invention to inhibitbeta7 integrin-ligand interaction to reduce or alleviate disease, suchas one or more of the above described disease states. In one embodiment,the antibody of the invention is used in the preparation of a medicamentfor the therapeutic and/or prophylactic treatment of a disease, such asan inflammatory disease including without limitation inflammatory boweldisease (such as Crohn's disease and ulcerative colitis), inflalmmatoryliver disease, inflammation of the CNS, chronic pancreatitis, systemiclupus erythematosus, Sjogren's syndrome, psoriasis and skininflammation, asthma, chronic obstructive pulmonary disease (COPD),interstitial lung disease, allergy, autoimmune disease, transplantationrejection, renal graft rejection, graft versus host disease, diabetes,and cancer.

In one aspect, the invention provides use of a nucleic acid of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as an immune (such asautoimmune or inflammatory) disorder including without limitation,inflammatory bowel disease (such as Crohn's disease or ulcerativecolitis) and allergic reaction (such as disorders of the respiratorysystem, skin, joints, allergic asthma and other organs affected byallergic reaction mediated by a beta7-containing integrin).

In one aspect, the invention provides use of an expression vector of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as an immune (such asautoimmune or inflammatory) disorder including without limitation,inflammatory bowel disease (such as Crohn's disease or ulcerativecolitis) and allergic reaction (such as disorders of the respiratorysystem, skin, joints, and other organs affected by allergic reactionmediated by a beta7-containing integrin).

In one aspect, the invention provides use of a host cell of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as an immune (such asautoimmune or inflammatory) disorder including without limitation,inflammatory bowel disease (such as Crohn's disease or ulcerativecolitis) and allergic reaction (such as disorders of the respiratorysystem, skin, joints, and other organs affected by allergic reactionmediated by a beta7-containing integrin).

In one aspect, the invention provides use of an article of manufactureof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disease, such as an immune (such asautoimmune or inflammatory) disorder including without limitation,inflammatory bowel disease (such as Crohn's disease or ulcerativecolitis) and allergic reaction (such as disorders of the respiratorysystem, skin, joints, and other organs affected by allergic reactionmediated by a beta7-containing integrin).

In one aspect, the invention provides use of a kit of the invention inthe preparation of a medicament for the therapeutic and/or prophylactictreatment of a disease, such as an immune (such as autoimmune orinflammatory) disorder including without limitation, inflammatory boweldisease (such as Crohn's disease or ulcerative colitis) and allergicreaction (such as disorders of the respiratory system, skin, joints, andother organs affected by allergic reaction mediated by abeta7-containing integrin).

The invention provides methods and compositions useful for modulatingdisease states associated with dysregulation of the beta7 integrinmediated cell-cell interaction process. The beta7 integrins are involvedin multiple biological and physiological functions, including, forexample, inflammatory disorders and allergic reactions. Thus, in oneaspect, the invention provides a method comprising administering to asubject an antibody of the invention.

In one aspect, the invention provides a method of inhibiting beta7integrin mediated inflammation, said method comprising contacting a cellor tissue with an effective amount of a antibody of the invention,whereby lymphocyte or B-cell interaction and binding to a beta7integrin-expressing cell is inhibited.

In one aspect, the invention provides a method of treating apathological condition associated with dysregulation of beta7 integrinbinding in a subject, said method comprising administering to thesubject an effective amount of an antibody of the invention, wherebysaid condition is treated.

In one aspect, the invention provides a method of inhibiting the bindingof a lymphocyte expressing a beta7 integrin ligand (such as a cellexpressing MAdCAM, VCAM, E-cadherein or fibronectin) to a cell thatexpresses beta7 integrin (such as alpha4beta7 or alphaEbeta7 integrins),said method comprising contacting said cell with an antibody of theinvention thereby inhibiting or preventing adhesion of the cells andcausing a reduction of inflammatory reaction.

In one aspect, the invention provides a method for treating orpreventing an inflammatory disorder associated with increased expressionor activity of beta7 integrin or increased interaction between a beta7integrin on one cell and a beta7 integrin receptor on another cell, saidmethod comprising administering to a subject in need of such treatmentan effective amount of an antibody of the invention, thereby effectivelytreating or preventing said inflammatory disorder. In one embodiment,said inflammatory disorder is inflammatory bowel disease (IBD). Inanother embodiment, said inflammatory disorder is an allergic reaction.

Methods of the invention can be used to affect any suitable pathologicalstate, for example, cells and/or tissues associated with dysregulationof the beta7 integrin binding pathway. Beta7 integrins are expressedprimarily on leukocytes (Tidswell, M. et al. (1997) supra). In oneembodiment, a leukocyte is targeted in a method of the invention and isprevented from binding to a cell expressing a ligand of the beta7integrin. For example, an an intra-epithelial lymphocyte expressingE-cadherin is prevented, according to the invention, from binding to analphaEbeta7-expressing cell by an antagonist anti-beta7 antibody. Cellsexpressing MAdCAM, VCAM-1 or fibronectin are prevented by an antagonistanti-beta7 antibody of the invention from binding to a leukocyteexpressing alpha4beta7.

Methods of the invention can further comprise additional treatmentsteps. For example, in one embodiment, a method further comprises a stepwherein a targeted cell and/or tissue (for example, an endothelial cellof the intestinal lining) is exposed to an anti-TNF antibody or a smallmolecule therapeutic agent including without limitation 5-ASA compounds(including without limitation

As described herein, beta7 integrins mediate important biologicalprocesses the dysregulation of which leads to numerous pathologicalconditions. Accordingly, in one embodiment of methods of the invention,a cell that is targeted (for example, an endothelial cell) is one inwhich adhesion of a cell expressing a beta7 integrin ligand of a beta7integrin (where the cell may be, without limitation, a lymphocyte, andthe ligand may be MAdCAM, VCAM or E-cadherin) is disrupted, inhibited,or prevented as compared to the cells in the absence of the anti-beta7antagonist antibody of the invention. In one embodiment, a method of theinvention inhibits lymphocyte homing, thereby inhibiting inflammation atthe site of beta7 integrin expression. For example, contact with anantagonist of the invention may result in a cell's inability to adhereto a cell expressing a ligand of a beta7 integrin .

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict alignment of sequences of the variable light andheavy chains for the following: light chain human subgroup kappa Iconsensus sequence (FIG. 1A, SEQ ID NO:23), heavy chain human subgroupIII consensus sequence (FIG. 1B, SEQ ID NO:24), rat anti-mouse beta7antibody (Fib504) variable light chain (FIG. 1A, SEQ ID NO:10), ratanti-mouse beta7 antibody (Fib504) variable heavy chain (FIG. 1B, SEQ IDNO:11),and humanized antibody vairiants: Humanized hu504Kgraft variablelight chain (FIG. 1A, SEQ ID NO:25), humanized hu504K graft variableheavy chain (FIG. 1B, SEQ ID NO:26), variant hu504.5 (amino acidvariations from humanized hu504K graft are indicated in FIG. 1A (lightchain) and FIG. 1B (heavy chain) for variants hu504.5, hu504.16, andhu504.32. Additioinal amino acid substitutions in the HVR-H1 and HVR-H2of the hu504K graft which resulted in beta7 binding antibodies areindicated in FIG. 1C.

FIGS. 2A and 2B depict the full length sequence of the human consensussubgroup III sequence light chain (FIG. 2A, SEQ ID NO:27) and heavychain (FIG. 2B, SEQ ID NO:28). HVRs are underlined.

FIGS. 3A and 3B depict the full length sequence of the humanized504graft containing rat Fib504 hypervariable regions (as described herein)grafted into the human kappa I consensus sequence light chain (FIG. 3A,SEQ ID NO:29) and into the human subgroup III consensus sequence heavychain (FIG. 3B, SEQ ID NO:30). HVRs are underlined.

FIGS. 4A and 4B depict the full length sequence of the humanized504Kgraft in which position 49 of the light chain of the hu504 graft isa Y49K substitution. The hu504Kgraft light chain is depicted by SEQ IDNO:31 and the hu504Kgraft heavy chain is depicted by SEQ ID NO:30. HVRsare underlined.

FIGS. 5A and 5B depict the full length sequence of the hu504K-RF graftin which positions 71 and 78 of the heavy chain of the hu504 graft arean A71R substitution and a A78F substitution from the hu504Kgraftsequence. The hu504K-RF graft light chain is depicted by SEQ ID NO:31and the hu504K-RF graft heavy chain is depicted by SEQ ID NO:32. HVRsare underlined.

FIGS. 6A and 6B depict the full length sequence of the hu504.32 variantcomprising the heavy chain of the hu504K-RF graft (SEQ ID NO:32) andT31D and Y32L substitutions in the light chain of the hu504Kgraft (SEQID NO:33). HVRs are underlined.

FIG. 7A-FIG. 7B and FIG. 8A-FIG. 8B depict exemplary acceptor humanconsensus framework sequences for use in practicing the instantinvention with sequence identifiers as follows:

Variable Light (VL) Consensus Frameworks (FIG. 7A,B)

-   human VL kappa subgroup I consensus framework (SEQ ID NO:14)-   human VL kappa subgroup I consensus framework minus extended HVR-L2    (SEQ ID NO:15)-   human VL kappa subgroup II consensus framework (SEQ ID NO:16)-   human VL kappa subgroup III consensus framework (SEQ ID NO:17)-   human VL kappa subgroup IV consensus framework (SEQ ID NO:18)-   Shaded regions represent light chain HVRs (indicated as L1, L2, and    L3).

Variable Heavy (VH) Consensus Frameworks (FIG. 8A, B)

-   human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID    NO:19)-   human VH subgroup I consensus framework minus extended hypervariable    regions (SEQ ID NOs:20-22)-   human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID    NO:48)-   human VH subgroup II consensus framework minus extended    hypervariable regions (SEQ ID NOs:49-51)-   human VH subgroup III consensus framework minus Kabat CDRs (SEQ ID    NO:52)-   human VH subgroup III consensus framework minus extended    hypervariable regions (SEQ ID NOs:53-55)-   human VH acceptor framework minus Kabat CDRs (SEQ ID NO:56)-   human VH acceptor framework minus extended hypervariable regions    (SEQ ID NOs:57-58)-   human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NO:59)-   human VH acceptor 2 framework minus extended hypervariable regions    (SEQ ID NOs: 60-62)

FIGS. 9A and 9B depict an amino acid sequence of the variable chains ofrat anti-mouse integrin beta7 Fib504 antibody produced by the hybridomaATCC HB-293. HVRs are underlined. Variable light chain is depicted inFIG. 9A (SEQ ID NO:12) and variable heavy chain is depicted in FIG. 9B(SEQ ID NO:13).

FIG. 10A depicts amino acid positions in the heavy chain of variousconsensus sequences (hu subgroups The consensus sequence used fordevelopment of the Herceptin® anti-HER2 antibody, rat Fib504, andhu504-RL and hu504-RF frameworks are described in the Examples herein.FIG. 10B is a bar graph showing the relative binding of alpha4beta7 tohu504graft antibody and hu504Kgraft antibody as a function of “RL” or“RF” framework modifications as described in Example 1.

FIG. 11A-11C. FIG. 11A tabulates the HVR changes resulting fromaffinity-maturation performed by offering a limited range of amino acidsubstitutions in the hu504.16 variant. The results are from librarieswith individually modified HVRs in the hu504.16 variant as described inExample 2 herein. Amino acid abbreviations in boxes are amino acidsfound more frequently in the beta7-binding antibodies (phage-selectedantibodies). FIGS. 11B and 11C are bar graphs of the results in FIG. 11Aindicating the number and type of amino acid substitutions in thehu504.16 variant (light chain, FIG. 11B; heavy chain, FIG. 11C)detectable by the mutagenesis and selection methods of Example 2.

FIG. 12 tabulates the results of affinity maturation performed byoffering a broad range of possible amino acid substitutions in the HVRsof hu504.32 variant as described in Example 2. The boxes indicate theamino acid that was detected most frequently in antibodies detected asbeta7-binding antibodies by the mutagenesis and selection methods ofExample 2.

FIGS. 13A and 13B depict HVR sequences of rat anti-mouse Fib504(ATCC-293), and the human consensus (left columns). Examples of aminoacid substitutions observed for each HVR position (not meant to belimiting) by the assays described in the Examples (amino acidsubstitutions observed by soft amino acid randomization, broad aminoacid substitution scan, and limited amino acid substitution scan) areshown to the right, (a useful method of modifying HVRs for humanization,applicable to variants of the present invention, is found in U.S.Application Ser. No. 60/545,840, filed Feb. 19, 2004).

FIG. 14 is an exemplary graphical representation of Fib504 and variantantibody binding to MAdCAM as a function of antibody concentration asdescribed in Example 3. IC₅₀ and IC₉₀ values for the antibodies weredetermined.

FIGS. 15A and 15B depict the light and heavy chain HVR amino acidsequences for the 504.32R anti-beta7 antibody with respect to positionaccording to the Kabat numbering system and a relative numbering system(A-F) for the six HVRs of the antibody. Amino acids at positions 71, 73,and 78 of the heavy chain FR3 region are also depicted. Useful aminoacid substitutions are also listed for many of the positions in the HVRsor the heavy chain FR3 region.

FIG. 16 shows bar graphs of the relative ability of the 504.32M and504.32R antibodies to block homing of radiolabelled T cells to the colonof mice experiencing inflammatory bowel disease.

MODES FOR CARRYING OUT THE INVENTION

The invention provides methods, compositions, kits and articles ofmanufacture for identifying and/or using inhibitors of the beta7signaling pathway.

Details of these methods, compositions, kits and articles of manufactureare provided herein.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., ed., 1994); “A Practical Guide to MolecularCloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual”(Barbas et al., 2001).

Definitions

By “beta7 subunit” or “(37 subunit” is meant the human β7 integrinsubunit (Erle et al., (1991) J. Biol. Chem. 266:11009-11016). The beta7subunit associates with alpha4 intetrin subunit, such as the human α4subunit (Kilger and Holzmann (1995) J. Mol. Biol. 73:347-354). Thealpha4beta7 integrin is expressed on a majority of mature lymphocytes,as well as a small population of thymocytes, bone marrow cells and mastcells. (Kilshaw and Murant (1991) Eur. J. Immunol. 21:2591-2597; Gurishet al., (1992) 149: 1964-1972; and Shaw, S. K. and Brenner, M. B. (1995)Semin. Immunol. 7:335). The beta7 subunit also associates with thealphaE subunit, such as the human alphaE integrin subunit (Cepek, K. L,et al. (1993) J. Immunol. 150:3459). The alphaEbeta7 integrin isexpressed on intra-intestinal epithelial lymphocytes (iIELs) (Cepek, K.L. (1993) supra). The beta7 subunit that binds to the humanizedanti-beta7 antibody of the invention may be naturally occurring and maybe soluble or localized to the surface of a cell.

By “alphaE subunit” or “alphaE integrin subunit” or “αE subunit” or “αEintegrin subunit” or “CD103” is meant an integrin subunit found to beassociated with beta7 integrin on intra-epithelial lymphocytes, whichalphaEbeta7 integrin mediates binding of the iELs to intestinalepithelium expressing E-caderin (Cepek, K. L. et al. (1993) J. Immunol.150:3459; Shaw, S. K. and Brenner, M. B. (1995) Semin. Immunol. 7:335).

“MAdCAM” or “MAdCAM-1” are used interchangeably in the context of thepresent invention and refer to the protein mucosal addressin celladhesion molecule-1, which is a single chain polypeptide comprising ashort cytoplasmic tail, a transmembrane region and an extracellularsequence composed of three immunoglobulin-like domains. The cDNAs formurine, human and macaque MAdCAM-1 have been cloned (Briskin, et al,(1993) Nature, 363:461-464; Shyjan et al., (1996) J. Immunol.156:2851-2857).

“VCAM-1” or “vascular cell adhesion molecule-1” “CD106” refers to aligand of alpha4beta7 and alpha4beta1, expressed on activatedendothelium and important in endothelial-leukocyte interactions such asbinding and transmigration of leukocytes during inflammation.

“E-cadherin” refers to a member of the family of cadherins, whereE-cadherin is expressed on epithelial cells. E-cadherin is a ligand ofthe alphaEbeta7 integrin and mediates binding of iEL-expressedalphaEbeta7 to intestinal epithelium, although its function inlymphocyte homing is unclear (.E-cadherin expression is upregulated byTGF-beta1.

“Fibronectin” refers to Fibronectin is involved in tissue repair,embryogenesis, blood clotting, and cell migration/adhesion. It serves asa linker in the ECM (extracellular matrix), and as dimer found in theplasma (plasma fibronectin). The plasma form is synthesized byhepatocytes, while the ECM form is made by fibroblasts, chondrocytes,endothelial cells, macrophages, as well as certain epithelial cells. Inthis context, it interacts with the alpha4beta7 integrin to mediateaspects of lymphocyte homing or adhesion. The ECM form of fibronectinserves as a general cell adhesion molecule by anchoring cells tocollagen or proteoglycan substrates. Fibronectin also can serve toorganize cellular interaction with the ECM by binding to differentcomponents of the extracellular matrix and to membrane-bound fibronectinreceptors on cell surfaces. Finally, fibronectin is important in cellmigration events during embryogenesis.

“Gastrointestinal inflammatory disorders” are a group of chronicdisorders that cause inflammation and/or ulceration in the mucousmembrane. These disorders include, for example, inflammatory boweldisease (e.g., Crohn's disease, ulcerative colitis, indeterminatecolitis and infectious colitis), mucositis (e.g., oral mucositis,gastrointestinal mucositis, nasal mucositis and proctitis), necrotizingenterocolitis and esophagitis.

“Inflammatory Bowel Disease” or “IBD” is used interchangeably herein torefer to diseases of the bowel that cause inflammation and/or ulcerationand includes without limitation Crohn's disease and ulcerative colitis.

“Crohn's disease (CD)” or “ulcerative colitis (UC)” are chronicinflammatory bowel diseases of unknown etiology. Crohn's disease, unlikeulcerative colitis, can affect any part of the bowel. The most prominentfeature Crohn's disease is the granular, reddish-purple edmatousthickening of the bowel wall. With the development of inflammation,these granulomas often lose their circumscribed borders and integratewith the surrounding tissue. Diarrhea and obstruction of the bowel arethe predominant clinical features. As with ulcerative colitis, thecourse of Crohn's disease may be continuous or relapsing, mild orsevere, but unlike ulcerative colitis, Crohn's disease is not curable byresection of the involved segment of bowel. Most patients with Crohn'sdisease require surgery at some point, but subsequent relapse is commonand continuous medical treatment is usual.

Crohn's disease may involve any part of the alimentary tract from themouth to the anus, although typically it appears in the ileocolic,small-intestinal or colonic-anorectal regions. Histopathologically, thedisease manifests by discontinuous granulomatomas, crypt abscesses,fissures and aphthous ulcers. The inflammatory infiltrate is mixed,consisting of lymphocytes (both T and B cells), plasma cells,macrophages, and neutrophils. There is a disproportionate increase inIgM- and IgG-secreting plasma cells, macrophages and neutrophils.

Anti-inflammatory drugs sulfasalazine and 5-aminosalisylic acid (5-ASA)are useful for treating mildly active colonic Crohn's disease and iscommonly perscribed to maintain remission of the disease. Metroidazoleand ciprofloxacin are similar in efficacy to sulfasalazine and appear tobe particularly useful for treating perianal disease. In more severecases, corticosteroids are effective in treating active exacerbationsand can even maintain remission. Azathioprine and 6-mercaptopurine havealso shown success in patients who require chronic administration ofcortico steroids. It is also possible that these drugs may play a rolein the long-term prophylaxis. Unfortunately, there can be a very longdelay (up to six months) before onset of action in some patients.

Antidiarrheal drugs can also provide symptomatic relief in somepatients. Nutritional therapy or elemental diet can improve thenutritional status of patients and induce symtomatic improvement ofacute disease, but it does not induce sustained clinical remissions.Antibiotics are used in treating secondary small bowel bacterialovergrowth and in treatment of pyogenic complications.

“Ulcerative colitis (UC)” afflicts the large intestine. The course ofthe disease may be continuous or relapsing, mild or severe. The earliestlesion is an inflammatory infiltration with abscess formation at thebase of the crypts of Lieberkühn. Coalescence of these distended andruptured crypts tends to separate the overlying mucosa from its bloodsupply, leading to ulceration. Symptoms of the disease include cramping,lower abdominal pain, rectal bleeding, and frequent, loose dischargesconsisting mainly of blood, pus and mucus with scanty fecal particles. Atotal colectomy may be required for acute, severe or chronic,unremitting ulcerative colitis.

The clinical features of UC are highly variable, and the onset may beinsidious or abrupt, and may include diarrhea, tenesmus and relapsingrectal bleeding. With fulminant involvement of the entire colon, toxicmegacolon, a life-threatening emergency, may occur. Extraintestinalmanifestations include arthritis, pyoderma gangrenoum, uveitis, anderythema nodosum.

Treatment for UC includes sulfasalazine and relatedsalicylate-containing drugs for mild cases and corticosteroid drugs insevere cases. Topical adminstration of either salicylates orcorticosteroids is sometimes effective, particularly when the disease islimited to the distal bowel, and is associated with decreased sideeffects compared with systemic use. Supportive measures such asadministration of iron and antidiarrheal agents are sometimes indicated.Azathioprine, 6-mercaptopurine and methotrexate are sometimes alsoprescribed for use in refractory corticosteroid-dependent cases.

A “modification” of an amino acid residue/position, as used herein,refers to a change of a primary amino acid sequence as compared to astarting amino acid sequence, wherein the change results from a sequencealteration involving said amino acid residue/positions. For example,typical modifications include substitution of the residue (or at saidposition) with another amino acid (e.g., a conservative ornon-conservative substitution), insertion of one or more (generallyfewer than 5 or 3) amino acids adjacent to said residue/position, anddeletion of said residue/position. An “amino acid substitution,” orvariation thereof, refers to the replacement of an existing amino acidresidue in a predetermined (starting) amino acid sequence with adifferent amino acid residue. Generally and preferably, the modificationresults in alteration in at least one physicobiochemical activity of thevariant polypeptide compared to a polypeptide comprising the starting(or “wild type”) amino acid sequence. For example, in the case of anantibody, a physicobiochemical activity that is altered can be bindingaffinity, binding capability and/or binding effect upon a targetmolecule.

The term “amino acid” within the scope of the present invention is usedin its broadest sense and is meant to include the naturally occurring Lα-amino acids or residues. The commonly used one- and three-letterabbreviations for naturally occurring amino acids are used herein(Lehninger, A. L., Biochemistry, 2d ed., pp. 71-92, (Worth Publishers,New York, N.Y., 1975). The term includes D-amino acids as well aschemically modified amino acids such as amino acid analogs, naturallyoccurring amino acids that are not usually incorporated into proteinssuch as norleucine, and chemically synthesized compounds havingproperties known in the art to be characteristic of an amino acid. Forexample, analogs or mimetics of phenylalanine or proline, which allowthe same conformational restriction of the peptide compounds as naturalPhe or Pro are included within the definition of amino acid. Suchanalogs and mimetics are referred to herein as “functional equivalents”of an amino acid. Other examples of amino acids are listed by Robertsand Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross andMeiehofer, Eds., Vol. 5, p. 341 (Academic Press, Inc., New York, N.Y.,1983), which is incorporated herein by reference. Where a single letteris used to designate one of the naturally occurring amino acid, thedesignations are as commonly found in the relevant literature (see, forexample, Alberts, B. et al. Molecular Biology of the Cell, 3rd ed.,Garland Publishing, Inc. 1994, page 57).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

The phrase “substantially similar,” or “substantially the same”, as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values is preferably less than about 50%, preferably less than about40%, preferably less than about 30%, preferably less than about 20%,preferably less than about 10% as a function of the value for thereference/comparator antibody.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay that measures solution binding affinity of Fabsfor antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol Biol 293:865-881). Toestablish conditions for the assay, microtiter plates (Dynex) are coatedovernight with 5 ug/ml of a capturing anti-Fab antibody (Cappel Labs) in50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)bovine serum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbant plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of an anti-VEGF antibody,Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., 65 hours) to insure that equilibriumis reached. Thereafter, the mixtures are transferred to the captureplate for incubation at room temperature (e.g., for one hour). Thesolution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 ul/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodimentthe Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25 C with immobilized antigen CMS chips at ˜10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CMS, BIAcoreInc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at a flow rate of 5ul/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 ul/min.Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol Biol 293:865-881. If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ bythe surface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25 C with immobilizedantigen CMS chips at ˜10 response units (RU). Briefly, carboxymethylateddextran biosensor chips (CMS, BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, into 5 ug/ml (˜0.2uM) before injection at a flow rate of 5 ul/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of 1M ethanolamine to block unreacted groups. For kineticsmeasurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) areinjected in PBS with 0.05% Tween 20 (PBST) at 25° C. at a flow rate ofapproximately 25 ul/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIAcore Evaluation Software version 3.2) by simultaneousfitting the association and dissociation sensorgram. The equilibriumdissociation constant (Kd) was calculated as the ratio k_(off)/k_(on).See, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881. However, ifthe on-rate exceeds 10⁶ M⁻¹ S⁻¹ by the surface plasmon resonance assayabove, then the on-rate is preferably determined by using a fluorescentquenching technique that measures the increase or decrease influorescence emission intensity (excitation=295 nm; emission=340 nm, 16nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) inPBS, pH 7.2, in the presence of increasing concentrations of antigen asmeasured in a a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-Amincospectrophotometer (ThermoSpectronic) with a stirred cuvette. The “Kd” or“Kd value” according to this invention is in one embodiment measured bya radiolabeled antigen binding assay (RIA) performed with the Fabversion of the antibody and antigen molecule as described by thefollowing assay that measures solution binding affinity of Fabs forantigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol Biol 293:865-881). Toestablish conditions for the assay, microtiter plates (Dynex) are coatedovernight with 5 ug/ml of a capturing anti-Fab antibody (Cappel Labs) in50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)bovine serum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbant plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (consistent with assessement of an anti-VEGF antibody, Fab-12,in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab of interestis then incubated overnight; however, the incubation may continue for alonger period (e.g., 65 hours) to insure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature for one hour. The solution is thenremoved and the plate washed eight times with 0.1% Tween-20 in PBS. Whenthe plates have dried, 150 ul/well of scintillant (MicroScint-20;Packard) is added, and the plates are counted on a Topcount gammacounter (Packard) for ten minutes. Concentrations of each Fab that giveless than or equal to 20% of maximal binding are chosen for use incompetitive binding assays. According to another embodiment, the Kd orKd value is measured by using surface plasmon resonance assays using aBIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25C with immobilized antigen CMS chips at ˜10 response units (RU).Briefly, carboxymethylated dextran biosensor chips (CMS, BIAcore Inc.)are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at a flow rate of 5ul/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 ul/min.Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol Biol 293:865-881. If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ bythe surface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

In one embodiment, an “on-rate” or “rate of association” or “associationrate” or “k_(on)” according to this invention is determined with thesame surface plasmon resonance technique described above using aBIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25C with immobilized antigen CMS chips at ˜10 response units (RU).Briefly, carboxymethylated dextran biosensor chips (CMS, BIAcore Inc.)are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at a flow rate of 5ul/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of 1M ethanolamine to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at25° C. at a flow rate of approximately 25 ul/min. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2) by simultaneous fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) was calculated asthe ratio k_(off)/k_(on). See, e.g., Chen, Y., et al., (1999) J. MolBiol 293:865-881. However, if the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by thesurface plasmon resonance assay above, then the on-rate is preferablydetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

The phrase “substantially reduced,” or “substantially different”, asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of statistical significance within the context ofthe biological characteristic measured by said values (e.g., Kd values,HAMA response). The difference between said two values is preferablygreater than about 10%, preferably greater than about 20%, preferablygreater than about 30%, preferably greater than about 40%, preferablygreater than about 50% as a function of the value for thereference/comparator antibody.

“Percent (%) amino acid sequence identity” with respect to a peptide orpolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximum ispercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2, wherein the complete source code for theALIGN-2 program is provided in Table A below. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc. and thesource code shown in Table A below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin FIG. 8 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional

DNA segments may be ligated. Another type of vector is a phage vector.Another type of vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “recombinant vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, .alpha.-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and abasic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S(“thioate”), P(S)S (“dithioate”), “(O)NR.sub.2 (“amidate”), P(O)R,P(O)OR′, CO or CH.sub.2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C.)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for example, fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multi specific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be human, humanized and/or affinitymatured.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. In one embodiment, an antibody fragment comprisesan antigen binding site of the intact antibody and thus retains theability to bind antigen. In another embodiment, an antibody fragment,for example one that comprises the Fc region, retains at least one ofthe biological functions normally associated with the Fc region whenpresent in an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise on antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Preferably, the target antigen is a polypeptide. An “acceptorhuman framework” for the purposes herein is a framework comprising theamino acid sequence of a VL or VH framework derived from a humanimmunoglobulin framework, or from a human consensus framework. Anacceptor human framework “derived from” a human immunoglobulin frameworkor human consensus framework may comprise the same amino acid sequencethereof, or may contain pre-existing amino acid sequence changes. Wherepre-existing amino acid changes are present, preferably no more than 5and preferably 4 or less, or 3 or less, pre-existing amino acid changesare present. Where pre-existing amino acid changes are present in a VH,preferably those changes are only at three, two or one of positions 71H,73H and 78H; for instance, the amino acid residues at those positionsmay be 71A, 73T and/or 78A. In one embodiment, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residue in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al. In one embodiment, for the VL, the subgroup is subgroupkappa I as in Kabat et al. In one embodiment, for the VH, the subgroupis subgroup III as in Kabat et al.

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al. In one embodiment, the VL subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

(SEQ ID NO: 34) DIQMTQSPSSLSASVGDRVTITC- (SEQ ID NO: 35)L1-WYQQKPGKAPKLLI- (SEQ ID NO: 36) L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC-(SEQ ID NO: 37) L3-FGQGTKVEIKR.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al. In one embodiment, the VH subgroup III consensus frameworkamino acid sequence comprises at least a portion or all of each of thefollowing sequences:

(SEQ ID NO: 38) EVQLVESGGGLVQPGGSLRLSCAAS- (SEQ ID NO: 39)H1-WVRQAPGKGLEWV- (SEQ ID NO: 40) H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA-(SEQ ID NO: 41) H3-WGQGTLVTVSS.

An “unmodified human framework” is a human framework which has the sameamino acid sequence as the acceptor human framework, e.g. lacking humanto non-human amino acid substitution(s) in the acceptor human framework.

An “altered hypervariable region” for the purposes herein is ahypervariable region comprising one or more (e.g. one to about 16) aminoacid substitution(s) therein.

An “un-modified hypervariable region” for the purposes herein is ahypervariable region having the same amino acid sequence as a non-humanantibody from which it was derived, i.e. one which lacks one or moreamino acid substitutions therein.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions are noted below.

TABLE 1 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B Kabat numbering H1 H31-H35H26-H35 H26-H32 H30-H35 Chothia numbering H2 H50-H65 H50-H58 H53-H55H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 49-56 or 50-56 or 52-56 (L2) and89-97 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102,94-102 or 95-102 (H3) in the VH. The variable domain residues arenumbered according to Kabat et al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it bind. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

A “disorder” is any condition that would benefit from treatment with asubstance/molecule or method of the invention. This includes chronic andacute disorders or diseases including those pathological conditionswhich predispose the mammal to the disorder in question. Non-limitingexamples of disorders to be treated herein include malignant and benigntumors; non-leukemias and lymphoid malignancies; neuronal, glial,astrocytal, hypothalamic and other glandular, macrophagal, epithelial,stromal and blastocoelic disorders; and inflammatory, immunologic andother angiogenesis-related disorders.

The term “immune related disease” means a disease in which a componentof the immune system of a mammal causes, mediates or otherwisecontributes to a morbidity in the mammal. Also included are diseases inwhich stimulation or intervention of the immune response has anameliorative effect on progression of the disease. Included within thisterm are immune-mediated inflammatory diseases, non-immune-mediatedinflammatory diseases, infectious diseases, immunodeficiency diseases,neoplasia, etc. Examples of immune-related and inflammatory diseases,some of which are immune or T cell mediated, which can be treatedaccording to the invention include systemic lupus erythematosis,rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies,systemic sclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease (ulcerative colitis: Crohn's disease),gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease. Infectious diseases includingviral diseases such as AIDS (HIV infection), hepatitis A, B, C, D, andE, herpes, etc., bacterial infections, fungal infections, protozoalinfections and parasitic infections.

An “autoimmune disorder” or “autoimmune disease” as used interchangeablyherein is a non-malignant disease or disorder arising from and directedagainst an individual's own tissues. The autoimmune diseases describedherein specifically exclude malignant or cancerous diseases orconditions, particularly excluding B cell lymphoma, acute lymphoblasticleukemia (ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemia,and chronic myeloblastic leukemia. Examples of autoimmune diseases ordisorders include, but are not limited to, inflammatory responses suchas inflammatory skin diseases including psoriasis and dermatitis (forexample, atopic dermatitis); systemic scleroderma and sclerosis;responses associated with inflammatory bowel disease (such as Crohn'sdisease and ulcerative colitis); respiratory distress syndrome(including adult respiratory distress syndrome; ARDS); dermatitis;meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergicconditions such as eczema and asthma and other conditions involvinginfiltration of T cells and chronic inflammatory responses;atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis;systemic lupus erythematosus (SLE); diabetes mellitus (e.g. Type Idiabetes mellitus or insulin dependent diabetes mellitis); multiplesclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergicencephalomyelitis; Sjorgen's syndrome; juvenile onset diabetes; andimmune responses associated with acute and delayed hypersensitivitymediated by cytokines and T-lymphocytes typically found in tuberculosis,sarcoidosis, polymyositis, granulomatosis and vasculitis; perniciousanemia (Addison's disease); diseases involving leukocyte diapedesis;central nervous system (CNS) inflammatory disorder; multiple organinjury syndrome; hemolytic anemia (including, but not limited tocryoglobinemia or Coombs positive anemia); myasthenia gravis;antigen-antibody complex mediated diseases; anti-glomerular basementmembrane disease; antiphospholipid syndrome; allergic neuritis; Graves'disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Behcet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia etc.

The term “gastrointestinal inflammatory disorders” is a group of chronicdisorders that cause inflammation and/or ulceration in themucousmembrane. As such, the term includes inflammatory bowel disease (e.g.,Crohn's disease, ulcerative colitis, indeterminate colitis andinfectious colitis), mucositis (e.g., oral mucositis, gastrointestinalmucositis, nasal mucositis and proctitis), necrotizing enterocolitis andesophagitis.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

Dysregulation of angiogenesis can lead to many disorders that can betreated by compositions and methods of the invention. These disordersinclude both non-neoplastic and neoplastic conditions. Neoplasticsinclude but are not limited those described above. Non-neoplasticdisorders include but are not limited to undesired or aberranthypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriaticplaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabeticand other proliferative retinopathies including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, chronic inflammation, lung inflammation, acutelung injury/ARDS, sepsis, primary pulmonary hypertension, malignantpulmonary effusions, cerebral edema (e.g., associated with acutestroke/closed head injury/trauma), synovial inflammation, pannusformation in RA, myositis ossificans, hypertropic bone formation,osteoarthritis (OA), refractory ascites, polycystic ovarian disease,endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartmentsyndrome, burns, bowel disease), uterine fibroids, premature labor,chronic inflammation such as IBD (Crohn's disease and ulcerativecolitis), renal allograft rejection, inflammatory bowel disease,nephrotic syndrome, undesired or aberrant tissue mass growth(non-cancer), hemophilic joints, hypertrophic scars, inhibition of hairgrowth, Osler-Weber syndrome, pyogenic granuloma retrolentalfibroplasias, scleroderma, trachoma, vascular adhesions, synovitis,dermatitis, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, antibodies of theinvention are used to delay development of a disease or disorder.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention, agonist or antagonist may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount will be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e. g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegall (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell whose growth is dependentupon beta7 activation either in vitro or in vivo. Thus, the growthinhibitory agent may be one which significantly reduces the percentageof beta7-dependent cells in S phase. Examples of growth inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (W B Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

Compounds useful in combination therapy with an antagonist anti-beta7antibody of the invention include antibodies (including withoutlimitation other anti-beta7 antagonist antibodies (Fib 21, 22, 27, 30(Tidswell, M. (1997) supra) or humanized derivatives thereof),anti-alpha4 antibodies (such as ANTEGEN®), anti-TNF (REMICADE®)) ornon-protein compounds including without limitation 5-ASA compoundsASACOL®, PENTASA™, ROWASA™, COLAZAL™, and other compounds such asPurinethol and steroids such as prednisone. In an embodiment, theinvention encompasses a method of treating a patient, such as a humanpatient, with the antagonist anti-beta7 antibody of the invention aloneor in combination with a second compound that is also useful in treatinginflammation. In one embodiment the second compound is selected from thegroup consisting of Fib 21, 22, 27, 30, or humanized derivativesthereof), anti-alpha4 antibodies, ANTEGEN®, anti-TNF, REMICADE®, 5-ASAcompounds, ASACOL®, PENTASA™, ROWASA™, COLAZAL™, Purinethol, steroids,and prednisone. In one embodiment of the invention, administration ofthe antagonist anti-beta7 antibody of the invention substantiallyreduces the dose of the second compound. In one embodiment, saidreduction in the dose of the second compound is at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%. In one embodiment of the invention, the combinationof the antibody of the invention and the reduced dose of the secondcompound relieves symptoms in the patient to substantially the samedegree or better than administration of the second compound alone.

Generating Variant Antibodies Exhibiting Reduced or Absence of HAMAResponse

Reduction or elimination of a HAMA (human anti-mouse (also applicable tohuman anti-rat or human anti-human) response is a significant aspect ofclinical development of suitable therapeutic agents. See, e.g.,Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al.,Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985),135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller etal., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352;Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res.(1990), 50:1495. As described herein, the invention provides antibodiesthat are humanized such that HAMA response is reduced or eliminated.Variants of these antibodies can further be obtained using routinemethods known in the art, some of which are further described below.

For example, an amino acid sequence from an antibody as described hereincan serve as a starting (parent) sequence for diversification of theframework and/or hypervariable sequence(s). A selected frameworksequence to which a starting hypervariable sequence is linked isreferred to herein as an acceptor human framework. While the acceptorhuman frameworks may be from, or derived from, a human immunoglobulin(the VL and/or VH regions thereof), preferably the acceptor humanframeworks are from, or derived from, a human consensus frameworksequence as such frameworks have been demonstrated to have minimal, orno, immunogenicity in human patients.

Where the acceptor is derived from a human immunoglobulin, one mayoptionally select a human framework sequence that is selected based onits homology to the donor framework sequence by aligning the donorframework sequence with various human framework sequences in acollection of human framework sequences, and select the most homologousframework sequence as the acceptor.

In one embodiment, human consensus frameworks herein are from, orderived from, VH subgroup III and/or VL kappa subgroup I consensusframework sequences.

Thus, the VH acceptor human framework may comprise one, two, three orall of the following framework sequences:

FR1 comprising (SEQ ID NO: 38) EVQLVESGGGLVQPGGSLRLSCAAS, FR2 comprising(SEQ ID NO: 39) WVRQAPGKGLEWV, FR3 comprising FR3 comprises(SEQ ID NO: 42) RFTISX1DX2SKNTX3YLQMNSLRAEDTAVYYCA, wherein X1 isA or R, X2 is T or N, and X3 is A, L, or F, FR4 comprising(SEQ ID NO: 41) WGQGTLVTVSS.Examples of VH consensus frameworks include:

-   human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID    NO:19);-   human VH subgroup I consensus framework minus extended hypervariable    regions (SEQ ID NOs:20-22);-   human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID    NO:48);-   human VH subgroup II consensus framework minus extended    hypervariable regions (SEQ ID NOs:49-51);-   human VH subgroup III consensus framework minus Kabat CDRs (SEQ ID    NO:52);-   human VH subgroup III consensus framework minus extended    hypervariable regions (SEQ ID NO:53-55);-   human VH acceptor framework minus Kabat CDRs (SEQ ID NO:56);-   human VH acceptor framework minus extended hypervariable regions    (SEQ ID NOs:57-58);-   human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NO:59); or-   human VH acceptor 2 framework minus extended hypervariable regions    (SEQ ID NOs:60-62).

In one embodiment, the VH acceptor human framework comprises one, two,three or all of the following framework sequences:

FR1 comprising (SEQ ID NO: 38) EVQLVESGGGLVQPGGSLRLSCAAS, FR2 comprising(SEQ ID NO: 39) WVRQAPGKGLEWV, FR3 comprising FR3 comprises(SEQ ID NO: 43) RFTISADTSKNTAYLQMNSLRAEDTAVYYCA, (SEQ ID NO: 44)RFTISRDTSKNTAYLQMNSLRAEDTAVYYCA, (SEQ ID NO: 45)RFTISRDTSKNTFYLQMNSLRAEDTAVYYCA, (SEQ ID NO: 46)RFTISADTSKNTFYLQMNSLRAEDTAVYYCA, FR4 comprising (SEQ ID NO: 41)WGQGTLVTVSS.

The VL acceptor human framework may comprise one, two, three or all ofthe following framework sequences:

FR1 comprising (SEQ ID NO: 34) DIQMTQSPSSLSASVGDRVTITC, FR2 comprising(SEQ ID NO: 35) WYQQKPGKAPKLLI, FR3 comprising (SEQ ID NO: 36)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC, FR4 comprising (SEQ ID NO: 37)FGQGTKVEIKR.Examples of VL consensus frameworks include:

-   human VL kappa subgroup I consensus framework (SEQ ID NO:14);-   human VL kappa subgroup I consensus framework (extended HVR-L2) (SEQ    ID NO:15);-   human VL kappa subgroup II consensus framework (SEQ ID NO:16);-   human VL kappa subgroup III consensus framework (SEQ ID NO:17); or-   human VL kappa subgroup IV consensus framework (SEQ ID NO:18)

While the acceptor may be identical in sequence to the human frameworksequence selected, whether that is from a human immunoglobulin or ahuman consensus framework, the present invention contemplates that theacceptor sequence may comprise pre-existing amino acid substitutionsrelative to the human immunoglobulin sequence or human consensusframework sequence. These pre-existing substitutions are preferablyminimal;

usually four, three, two or one amino acid differences only relative tothe human immunoglobulin sequence or consensus framework sequence.

Hypervariable region residues of the non-human antibody are incorporatedinto the VL and/or VH acceptor human frameworks. For example, one mayincorporate residues corresponding to the Kabat CDR residues, theChothia hypervariable loop residues, the

Abm residues, and/or contact residues. Optionally, the extendedhypervariable region residues as follows are incorporated: 24-34 (L1),49-56 (L2) and 89-97 (L3), 26-35 (H1), 50-65 or 49-65 (H2) and 93-102,94-102, or 95-102 (H3).

While “incorporation” of hypervariable region residues is discussedherein, it will be appreciated that this can be achieved in variousways, for example, nucleic acid encoding the desired amino acid sequencecan be generated by mutating nucleic acid encoding the mouse variabledomain sequence so that the framework residues thereof are changed toacceptor human framework residues, or by mutating nucleic acid encodingthe human variable domain sequence so that the hypervariable domainresidues are changed to non-human residues, or by synthesizing nucleicacid encoding the desired sequence, etc.

In the examples herein, hypervariable region-grafted variants weregenerated by Kunkel mutagenesis of nucleic acid encoding the humanacceptor sequences, using a separate oligonucleotide for eachhypervariable region. Kunkel et al., Methods Enzymol. 154:367-382(1987). Appropriate changes can be introduced within the frameworkand/or hypervariable region, using routine techniques, to correct andre-establish proper hypervariable region-antigen interactions.

Phage(mid) display (also referred to herein as phage display in somecontexts) can be used as a convenient and fast method for generating andscreening many different potential variant antibodies in a librarygenerated by sequence randomization. However, other methods for makingand screening altered antibodies are available to the skilled person.

Phage(mid) display technology has provided a powerful tool forgenerating and selecting novel proteins which bind to a ligand, such asan antigen. Using the techniques of phage(mid) display allows thegeneration of large libraries of protein variants which can be rapidlysorted for those sequences that bind to a target molecule with highaffinity. Nucleic acids encoding variant polypeptides are generallyfused to a nucleic acid sequence encoding a viral coat protein, such asthe gene III protein or the gene VIII protein. Monovalent phagemiddisplay systems where the nucleic acid sequence encoding the protein orpolypeptide is fused to a nucleic acid sequence encoding a portion ofthe gene III protein have been developed. (Bass, S., Proteins, 8:309(1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology,3:205 (1991)). In a monovalent phagemid display system, the gene fusionis expressed at low levels and wild type gene III proteins are alsoexpressed so that infectivity of the particles is retained. Methods ofgenerating peptide libraries and screening those libraries have beendisclosed in many patents (e.g. U.S. Pat. No. 5,723,286, U.S. Pat. No.5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908 and U.S.Pat. No. 5,498,530).

Libraries of antibodies or antigen binding polypeptides have beenprepared in a number of ways including by altering a single gene byinserting random DNA sequences or by cloning a family of related genes.Methods for displaying antibodies or antigen binding fragments usingphage(mid) display have been described in U.S. Pat. Nos. 5,750,373,5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727.The library is then screened for expression of antibodies or antigenbinding proteins with the desired characteristics.

Methods of substituting an amino acid of choice into a template nucleicacid are well established in the art, some of which are describedherein. For example, hypervariable region residues can be substitutedusing the Kunkel method. See, for example, Kunkel et al., MethodsEnzymol. 154:367-382 (1987).

The sequence of oligonucleotides includes one or more of the designedcodon sets for the hypervariable region residues to be altered. A codonset is a set of different nucleotide triplet sequences used to encodedesired variant amino acids. Codon sets can be represented using symbolsto designate particular nucleotides or equimolar mixtures of nucleotidesas shown in below according to the IUB code.

IUB Codes

G Guanine

A Adenine

T Thymine

C Cytosine

R (A or G)

Y (C or T)

M (A or C)

K (G or T)

S (C or G)

W (A or T)

H (A or C or T)

B (C or G or T)

V (A or C or G)

D (A or G or T) H

N (A or C or G or T)

For example, in the codon set DVK, D can be nucleotides A or G or T; Vcan be A or G or C; and K can be G or T. This codon set can present 18different codons and can encode amino acids Ala, Trp, Tyr, Lys, Thr,Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

Oligonucleotide or primer sets can be synthesized using standardmethods. A set of oligonucleotides can be synthesized, for example, bysolid phase synthesis, containing sequences that represent all possiblecombinations of nucleotide triplets provided by the codon set and thatwill encode the desired group of amino acids. Synthesis ofoligonucleotides with selected nucleotide “degeneracy” at certainpositions is well known in that art. Such sets of nucleotides havingcertain codon sets can be synthesized using commercial nucleic acidsynthesizers (available from, for example, Applied Biosystems, FosterCity, Calif.), or can be obtained commercially (for example, from LifeTechnologies, Rockville, Md.). Therefore, a set of oligonucleotidessynthesized having a particular codon set will typically include aplurality of oligonucleotides with different sequences, the differencesestablished by the codon set within the overall sequence.Oligonucleotides, as used according to the invention, have sequencesthat allow for hybridization to a variable domain nucleic acid templateand also can include restriction enzyme sites for cloning purposes.

In one method, nucleic acid sequences encoding variant amino acids canbe created by oligonucleotide-mediated mutagenesis. This technique iswell known in the art as described by Zoller et al. Nucleic Acids Res.10:6487-6504(1987). Briefly, nucleic acid sequences encoding variantamino acids are created by hybridizing an oligonucleotide set encodingthe desired codon sets to a DNA template, where the template is thesingle-stranded form of the plasmid containing a variable region nucleicacid template sequence. After hybridization, DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will contain thecodon sets as provided by the oligonucleotide set.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation(s). This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Nat'l. Acad. Sci. USA, 75:5765 (1978).

The DNA template is generated by those vectors that are either derivedfrom bacteriophage M13 vectors (the commercially available M13mp18 andM13mp19 vectors are suitable), or those vectors that contain asingle-stranded phage origin of replication as described by Viera etal., Meth. Enzymol., 153:3 (1987). Thus, the DNA that is to be mutatedcan be inserted into one of these vectors in order to generatesingle-stranded template. Production of the single-stranded template isdescribed in sections 4.21-4.41 of Sambrook et al., above.

To alter the native DNA sequence, the oligonucleotide is hybridized tothe single stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually T7 DNA polymerase or the Klenowfragment of DNA polymerase I, is then added to synthesize thecomplementary strand of the template using the oligonucleotide as aprimer for synthesis. A heteroduplex molecule is thus formed such thatone strand of DNA encodes the mutated form of gene 1, and the otherstrand (the original template) encodes the native, unaltered sequence ofgene 1. This heteroduplex molecule is then transformed into a suitablehost cell, usually a prokaryote such as E. coli JM101. After growing thecells, they are plated onto agarose plates and screened using theoligonucleotide primer radiolabelled with a 32-Phosphate to identify thebacterial colonies that contain the mutated DNA.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: The singlestranded oligonucleotide is annealed to the single-stranded template asdescribed above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTT), is combined with a modifiedthiodeoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham). This mixture is added to the template-oligonucleotidecomplex. Upon addition of DNA polymerase to this mixture, a strand ofDNA identical to the template except for the mutated bases is generated.In addition, this new strand of DNA will contain dCTP-(aS) instead ofdCTP, which serves to protect it from restriction endonucleasedigestion. After the template strand of the double-stranded heteroduplexis nicked with an appropriate restriction enzyme, the template strandcan be digested with ExoIII nuclease or another appropriate nucleasepast the region that contains the site(s) to be mutagenized. Thereaction is then stopped to leave a molecule that is only partiallysingle-stranded. A complete double-stranded DNA homoduplex is thenformed using DNA polymerase in the presence of all fourdeoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplexmolecule can then be transformed into a suitable host cell.

As indicated previously the sequence of the oligonucleotide set is ofsufficient length to hybridize to the template nucleic acid and mayalso, but does not necessarily, contain restriction sites. The DNAtemplate can be generated by those vectors that are either derived frombacteriophage M13 vectors or vectors that contain a single-strandedphage origin of replication as described by Viera et al. Meth. Enzymol.,153:3 (1987). Thus, the DNA that is to be mutated must be inserted intoone of these vectors in order to generate single-stranded template.Production of the single-stranded template is described in sections4.21-4.41 of Sambrook et al., supra.

According to another method, a library can be generated by providingupstream and downstream oligonucleotide sets, each set having aplurality of oligonucleotides with different sequences, the differentsequences established by the codon sets provided within the sequence ofthe oligonucleotides. The upstream and downstream oligonucleotide sets,along with a variable domain template nucleic acid sequence, can be usedin a polymerase chain reaction to generate a “library” of PCR products.The PCR products can be referred to as “nucleic acid cassettes”, as theycan be fused with other related or unrelated nucleic acid sequences, forexample, viral coat proteins and dimerization domains, using establishedmolecular biology techniques.

The sequence of the PCR primers includes one or more of the designedcodon sets for the solvent accessible and highly diverse positions in ahypervariable region. As described above, a codon set is a set ofdifferent nucleotide triplet sequences used to encode desired variantamino acids.

Antibody selectants that meet the desired criteria, as selected throughappropriate screening/selection steps can be isolated and cloned usingstandard recombinant techniques.

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, preferredhost cells are of either prokaryotic or eukaryotic (generally mammalian)origin.

Generating Antibodies Using Prokaryotic Host Cells: Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated is regulatory sequence located upstream (5′)to a cistron that modulates its expression.

Prokaryotic promoters typically fall into two classes, inducible andconstitutive. Inducible promoter is a promoter that initiates increasedlevels of transcription of the cistron under its control in response tochanges in the culture condition, e.g. the presence or absence of anutrient or a change in temperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. coli trxB⁻strains) provide cytoplasm conditions that are favorable for disulfidebond formation, thereby permitting proper folding and assembly ofexpressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

The present invention provides an expression system in which thequantitative ratio of expressed polypeptide components can be modulatedin order to maximize the yield of secreted and properly assembledantibodies of the invention. Such modulation is accomplished at least inpart by simultaneously modulating translational strengths for thepolypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence, although silent changes in the nucleotide sequenceare preferred. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

Preferably, a set of vectors is generated with a range of TIR strengthsfor each cistron therein. This limited set provides a comparison ofexpression levels of each chain as well as the yield of the desiredantibody products under various TIR strength combinations. TIR strengthscan be determined by quantifying the expression level of a reporter geneas described in detail in Simmons et al. U.S. Pat. No. 5,840,523. Basedon the translational strength comparison, the desired individual TIRsare selected to be combined in the expression vector constructs of theinvention.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms.

Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli(e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P.aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella,Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In oneembodiment, gram-negative cells are used. In one embodiment, E. colicells are used as hosts for the invention. Examples of E. coli strainsinclude strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2(Washington, D.C.: American Society for Microbiology, 1987), pp.1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, includingstrain 33D3 having genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE) degP41 kan^(R) (U.S. Pat. No. 5,639,635). Otherstrains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E.coli B, E. coli _(λ) 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608)are also suitable. These examples are illustrative rather than limiting.Methods for constructing derivatives of any of the above-mentionedbacteria having defined genotypes are known in the art and described in,for example, Bass et al., Proteins, 8:309-314 (1990). It is generallynecessary to select the appropriate bacteria taking into considerationreplicability of the replicon in the cells of a bacterium. For example,E. coli, Serratia, or Salmonella species can be suitably used as thehost when well known plasmids such as pBR322, pBR325, pACYC177, orpKN410 are used to supply the replicon. Typically the host cell shouldsecrete minimal amounts of proteolytic enzymes, and additional proteaseinhibitors may desirably be incorporated in the cell culture.

Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some is embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, for example,Simmons et al., J. Immunol. Methods (2002), 263:133-147). A variety ofother inducers may be used, according to the vector construct employed,as is known in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay. In one aspect of the invention, antibodyproduction is conducted in large quantity by a fermentation process.Various large-scale fed-batch fermentation procedures are available forproduction of recombinant proteins. Large-scale fermentations have atleast 1000 liters of capacity, preferably about 1,000 to 100,000 litersof capacity. These fermentors use agitator impellers to distributeoxygen and nutrients, especially glucose (the preferred carbon/energysource). Small scale fermentation refers generally to fermentation in afermentor that is no more than approximately 100 liters in volumetriccapacity, and can range from about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD₅₅₀ of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

Antibody Purification

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products of theinvention. Protein A is a 41 kD cell wall protein from Staphylococcusaureas which binds with a high affinity to the Fc region of antibodies.Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase towhich Protein A is immobilized is preferably a column comprising a glassor silica surface, more preferably a controlled pore glass column or asilicic acid column. In some applications, the column has been coatedwith a reagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

Generating Antibodies Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma-Aldrich, St. Louis, Mo., USA), Minimal Essential Medium((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ((DMEM), Sigma) are suitable for culturing the host cells. Inaddition, any of the media described in Ham et al., Meth. Enz. 58:44(1979), Barnes et al., Anal. Biochem.102:255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430;WO 87/00195; or U.S. Patent Re. 30,985 may be used as culture media forthe host cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleotides (such asadenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Activity Assays

The antibodies of the present invention can be characterized for theirphysical/chemical properties and biological functions by various assaysknown in the art.

The purified immunoglobulins can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the immunoglobulins producedherein are analyzed for their biological activity. In some embodiments,the immunoglobulins of the present invention are tested for theirantigen binding activity. The antigen binding assays that are known inthe art and can be used herein include without limitation any direct orcompetitive binding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immnosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays. An illustrative antigen binding assay isprovided below in the Examples section.

In one embodiment, the present invention contemplates an alteredantibody that possesses some but not all effector functions, which makeit a desired candidate for many applications in which the half life ofthe antibody in vivo is important yet certain effector functions (suchas complement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the produced immunoglobulin aremeasured to ensure that only the desired properties are maintained. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Anexample of an in vitro assay to assess ADCC activity of a molecule ofinterest is described in U.S. Pat. Nos. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.PNAS (USA) 95:652-656 (1998). C1q binding assays may also be carried outto confirm that the antibody is unable to bind C1q and hence lacks CDCactivity. To assess complement activation, a CDC assay, for example asdescribed in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996),may be performed. FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art, forexample those desribed in the Examples section.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

Antibody Variants

In one aspect, the invention provides antibody fragment comprisingmodifications in the interface of Fc polypeptides comprising the Fcregion, wherein the modifications facilitate and/or promoteheterodimerization. These modifications comprise introduction of aprotuberance into a first Fc polypeptide and a cavity into a second Fcpolypeptide, wherein the protuberance is positionable in the cavity soas to promote complexing of the first and second Fc polypeptides.Methods of generating antibodies with these modifications are known inthe art, for example, as described in U.S. Pat. No. 5,731,168.

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid alterations may beintroduced in the subject antibody amino acid sequence at the time thatsequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 2 under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 2,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE 2 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)):

-   (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F),    Trp (W), Met (M)-   (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y),    Asn (N), Gln (Q)-   (3) acidic: Asp (D), Glu (E)-   (4) basic: Lys (K), Arg (R), His(H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibodies thus generated are displayed fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides of the invention,thereby generating a Fc region variant. The Fc region variant maycomprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 orIgG4 Fc region) comprising an amino acid modification (e.g. asubstitution) at one or more amino acid positions including that of ahinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody used in methods ofthe invention may comprise one or more alterations as compared to thewild type counterpart antibody, for example in the Fc region. Theseantibodies would nonetheless retain substantially the samecharacteristics required for therapeutic utility as compared to theirwild type counterpart. For example, it is thought that certainalterations can be made in the Fc region that would result in altered(i.e., either improved or diminished) C1q binding and/or ComplementDependent Cytotoxicity (CDC), for example, as described in WO99/51642.See also Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO94/29351 concerning otherexamples of Fc region variants.

Immunoconjugates

The invention also pertains to immunoconjugates, or antibody-drugconjugates (ADC), comprising an antibody conjugated to a cytotoxic agentsuch as a chemotherapeutic agent, a drug, a growth inhibitory agent, atoxin (e.g., an enzymatically active toxin of bacterial, fungal, plant,or animal origin, or fragments thereof), or a radioactive isotope (i.e.,a radioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) theoretically allows targeteddelivery of the drug moiety to tumors, and intracellular accumulationtherein, where systemic administration of these unconjugated drug agentsmay result in unacceptable levels of toxicity to normal cells as well asthe tumor cells sought to be eliminated (Baldwin et al., (1986) Lancetpp. (Mar. 15, 1986):603-05; Thorpe, (1985) “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies'84: Biological And Clinical Applications, A. Pinchera et al. (ed.s),pp. 475-506). Maximal efficacy with minimal toxicity is sought thereby.Both polyclonal antibodies and monoclonal antibodies have been reportedas useful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Thetoxins may effect their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of an anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the protential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE),monomethylauristatin E (MMAE), and synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al. (2003) Nature Biotechnology21(7):778-784; and Francisco, et al. (2003) Blood, 102, 1458-1465) andare under therapeutic development. Other compounds for use as drugconjugate cytotoxic agents include without limitation auristatin E (AE),MMAF (a variant of auristatin E (MMAE) with a phenylalanine at theC-terminus of the drug), and AEVB (auristatin E valeryl benzylhydrazone,an acid labile linker through the C-terminus of AE). Useful conjugatelinkers for attaching a drug to an antibody include without limitationMC (maleimidocaproyl), Val Cit (valine-citrulline, dipeptide site inprotease cleavable linker), Citrulline (2-amino-5-ureido pentanoicacid), PAB (p-aminobenzylcarbamoyl, a “self immolative” portion oflinker), Me (N-methyl-valine citrulline where the linker peptide bondhas been modified to prevent its cleavage by cathepsin B), MC(PEG)6-OH(maleimidocaproyl-polyethylene glycol, attached to antibody cysteines),SPP (N-Succinimidyl 4-(2-pyridylthio) pentanoate), and SMCC(N-Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate). Theseand other useful drug conjugates and their preparation are disclosed,for example, in Doronina, S. O. et al., Nature Biotechnology 21:778-794(2003), incorporated herein by reference in its entirety. Particularlypreferred linker molecules include, for example, N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) (see, e.g., Carlsson et al.,Biochem. J., 173, 723-737 (1978)), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No.4,563,304), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (see,e.g., CAS Registry number 341498-08-6), N-succinimidyl4-(N-maleimidomethyl)cyclohe-xane-1-carboxylate (SMCC) (see, e.g.,Yoshitake et al., Eur. J. Biochem., 101, 395-399 (1979)), andN-succinimidyl 4-methyl-4-[2-(5-nitro-pyridyl)-dithio]pentanoate (SMNP)(see, e.g., U.S. Pat. No. 4,563,304).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, a trichothecene, and CC1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

Maytansine and Maytansinoids

In one embodiment, an antibody (full length or fragments) of theinvention is conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1, the disclosures of whichare hereby expressly incorporated by reference. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DM1 linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al.,Cancer Research 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10⁵ HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Antibody-Maytansinoid Conjugates (Immunoconjugates)

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. An average of 3-4 maytansinoid molecules conjugated perantibody molecule has shown efficacy in enhancing cytotoxicity of targetcells without negatively affecting the function or solubility of theantibody, although even one molecule of toxin/antibody would be expectedto enhance cytotoxicity over the use of naked antibody. Maytansinoidsare well known in the art and can be synthesized by known techniques orisolated from natural sources. Suitable maytansinoids are disclosed, forexample, in U.S. Pat. No. 5,208,020 and in the other patents andnonpatent publications referred to hereinabove. Preferred maytansinoidsare maytansinol and maytansinol analogues modified in the aromatic ringor at other positions of the maytansinol molecule, such as variousmaytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et al., Cancer Research 52:127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

Calicheamicin

Another immunoconjugate of interest comprises an antibody conjugated toone or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. For the preparation of conjugates of thecalicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all toAmerican Cyanamid Company). Structural analogues of calicheamicin whichmay be used include, but are not limited to, γ₁ ^(I), α₁ ^(I), α₃ ^(I),N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (Hinman et al., Cancer Research53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998)and the aforementioned U.S. patents to American Cyanamid). Anotheranti-tumor drug that the antibody can be conjugated is QFA which is anantifolate. Both calicheamicin and QFA have intracellular sites ofaction and do not readily cross the plasma membrane. Therefore, cellularuptake of these agents through antibody mediated internalization greatlyenhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, .Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinyl sulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC) of the invention, an antibody (Ab)is conjugated to one or more drug moieties (D), e.g. about 1 to about 20drug moieties per antibody, through a linker (L). The ADC of Formula Imay be prepared by several routes, employing organic chemistryreactions, conditions, and reagents known to those skilled in the art,including: (1) reaction of a nucleophilic group of an antibody with abivalent linker reagent, to form Ab-L, via a covalent bond, followed byreaction with a drug moiety D; and (2) reaction of a nucleophilic groupof a drug moiety with a bivalent linker reagent, to form D-L, via acovalent bond, followed by reaction with the nucleophilic group of anantibody.

Ab-(L-D)_(p)   I

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol.

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic subsituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g. withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g. by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either glactose oxidase or sodium meta-periodate may yieldcarbonyl (aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymers are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody of the invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Uses

An antibody of the present invention may be used in, for example, invitro, ex vivo and in vivo therapeutic methods. Antibodies of theinvention can be used as an antagonist to partially or fully block thespecific antigen activity in vitro, ex vivo and/or in vivo. Moreover, atleast some of the antibodies of the invention can neutralize antigenactivity from other species. Accordingly, the antibodies of theinvention can be used to inhibit a specific antigen activity, e.g., in acell culture containing the antigen, in human subjects or in othermammalian subjects having the antigen with which an antibody of theinvention cross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgusand rhesus, pig or mouse). In one embodiment, the antibody of theinvention can be used for inhibiting antigen activities by contactingthe antibody with the antigen such that antigen activity is inhibited.Preferably, the antigen is a human protein molecule.

In one embodiment, an antibody of the invention can be used in a methodfor inhibiting an antigen in a subject suffering from a disorder inwhich the antigen activity is detrimental, comprising administering tothe subject an antibody of the invention such that the antigen activityin the subject is inhibited. Preferably, the antigen is a human proteinmolecule and the subject is a human subject. Alternatively, the subjectcan be a mammal expressing the antigen with which an antibody of theinvention binds. Still further the subject can be a mammal into whichthe antigen has been introduced (e.g., by administration of the antigenor by expression of an antigen transgene). An antibody of the inventioncan be administered to a human subject for therapeutic purposes.Moreover, an antibody of the invention can be administered to anon-human mammal expressing an antigen with which the immunoglobulincross-reacts (e.g., a primate, pig or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration). Blocking antibodies of the invention that aretherapeutically useful include, for example but are not limited to,anti-HER2, anti-VEGF, anti-IgE, anti-CD11, anti-interferon andanti-tissue factor antibodies. The antibodies of the invention can beused to treat, inhibit, delay progression of, prevent/delay recurrenceof, ameliorate, or prevent diseases, disorders or conditions associatedwith abnormal expression and/or activity of one or more antigenmolecules, including but not limited to malignant and benign tumors;non-leukemias and lymphoid malignancies; neuronal, glial, astrocytal,hypothalamic and other glandular, macrophagal, epithelial, stromal andblastocoelic disorders; and inflammatory, angiogenic and immunologicdisorders.

In one aspect, a blocking antibody of the invention is specific to aligand antigen, and inhibits the antigen activity by blocking orinterfering with the ligand-receptor interaction involving the ligandantigen, thereby inhibiting the corresponding signal pathway and othermolecular or cellular events. The invention also featuresreceptor-specific antibodies which do not necessarily prevent ligandbinding but interfere with receptor activation, thereby inhibiting anyresponses that would normally be initiated by the ligand binding. Theinvention also encompasses antibodies that either preferably orexclusively bind to ligand-receptor complexes. An antibody of theinvention can also act as an agonist of a particular antigen receptor,thereby potentiating, enhancing or activating either all or partialactivities of the ligand-mediated receptor activation.

In certain embodiments, an immunoconjugate comprising an antibodyconjugated with a cytotoxic agent is administered to the patient. Insome embodiments, the immunoconjugate and/or antigen to which it isbound is/are internalized by the cell, resulting in increasedtherapeutic efficacy of the immunoconjugate in killing the target cellto which it binds. In one embodiment, the cytotoxic agent targets orinterferes with nucleic acid in the target cell. Examples of suchcytotoxic agents include any of the chemotherapeutic agents noted herein(such as a maytansinoid or a calicheamicin), a radioactive isotope, or aribonuclease or a DNA endonuclease.

Antibodies of the invention can be used either alone or in combinationwith other compositions in a therapy. For instance, an antibody of theinvention may be co-administered with another antibody, chemotherapeuticagent(s) (including cocktails of chemotherapeutic agents), othercytotoxic agent(s), anti-angiogenic agent(s), cytokines, and/or growthinhibitory agent(s). Where an antibody of the invention inhibits tumorgrowth, it may be particularly desirable to combine it with one or moreother therapeutic agent(s) which also inhibits tumor growth. Forinstance, an antibody of the invention may be combined with an anti-VEGFantibody (e.g., AVASTIN) and/or anti-ErbB antibodies (e.g. HERCEPTIN®anti-HER2 antibody) in a treatment scheme, e.g. in treating any of thediseases described herein, including colorectal cancer, metastaticbreast cancer and kidney cancer. Alternatively, or additionally, thepatient may receive combined radiation therapy (e.g. external beamirradiation or therapy with a radioactive labeled agent, such as anantibody). Such combined therapies noted above include combinedadministration (where the two or more agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,and/or following, administration of the adjunct therapy or therapies.

The antibody of the invention (and adjunct therapeutic agent) is/areadministered by any suitable means, including parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody is suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Dosing can be by any suitable route, for example byinjections, such as intravenous or subcutaneous injections, depending inpart on whether the administration is brief or chronic.

The antibody composition of the invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount ofantibodies of the invention present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with otheragents such as chemotherapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody is an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. One exemplary dosage of theantibody would be in the range from about 0.05 mg/kg to about 10 mg/kg.Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient.Such doses may be administered intermittently, e.g. every week or everythree weeks (e.g. such that the patient receives from about two to abouttwenty, e.g. about six doses of the antibody). An initial higher loadingdose, followed by one or more lower doses may be administered. Anexemplary dosing regimen comprises administering an initial loading doseof about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kgof the antibody. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or when combined with another composition effectivefor treating, preventing and/or diagnosing the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anantibody of the invention. The label or package insert indicates thatthe composition is used for treating the condition of choice, such ascancer. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic agent. The article of manufacture in this embodimentof the invention may further comprise a package insert indicating thatthe first and second antibody compositions can be used to treat aparticular condition, for example cancer. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

EXAMPLES

The examples herein describe the generation of humanized anti-beta7antibodies from a rat anti-mouse antibody that binds to the beta7subunit of the alpha4beta7 integrin.

Example 1 Humanization of a Beta7 Antagonist Antibody

Materials and Methods

Residue numbers are according to Kabat (Kabat et al., Sequences ofproteins of immunological interest, 5th Ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Single letteramino acid abbreviations are used. DNA degeneracies are representedusing the IUB code (N=A/C/G/T, D=A/G/T, V=A/C/G, B=C/G/T, H=A/C/T,K=G/T, M=A/C, R=A/G, S=G/C, W=A/T, Y=C/T).

Direct hypervariable region grafts onto the acceptor human consensusframework—The phagemid used for this work, pVO350-2b, was a monovalentFab-g3 display vector having 2 open reading frames under control of thephoA promoter, essentially as described in Lee et al., J. Mol. Biol.(2004), 340(5):1073-93. The first open reading frame consists of thestII signal sequence fused to the VL and CH1 domains of the acceptorlight chain and the second consists of the stII signal sequence fused tothe VH and CH1 domains of the acceptor heavy chain followed by atruncated form of the minor phage coat protein P3 (Lowman, H. et al.(1990) Biochemistry 30:10832).

The VL and VH domains from rat Fib504 (antibody FM504.64 produced byhybridoma ATCC HB-293, American Type Culture Collection (ATCC), P.O. Box1549, Manassas, Va. 20108, USA) were aligned with the human consensuskappa I (huKI) and human subgroup III consensus VH (huIII) domains. Tomake the hypervariable region (HVR) grafts, the following frameworkswere used: HuKI was used for the light chain variable domain framework(see FIGS. 1A and 7). For the heavy chain variable domain framework, theacceptor VH framework, a modified human subgroup III (humIII) consensusVH domain which differs from the humIII sequence at 3 positions: R71A,N73T, and L78A may be used (see Carter et al., Proc. Natl. Acad. Sci.USA 89:4285 (1992)) (see FIG. 1B). In generation of antibodies of thepresent invention, the 504K-RF graft was also prepared from the modifiedhuman subgroupIII consensus VH domain by making the following amino acidsubstitutions: A71R and A78F.

Hypervariable regions from rat Fib504 (produced by hybridoma ATCCHB-293) antibody were engineered into the acceptor human subgroup IIIconsensus VH framework to generate a direct HVR-graft (Fib504graft) (seeFIG. 1B). In the VL domain the following regions from rat Fib504 weregrafted to the human consensus acceptor, huKI: positions 24-34 (L1),50-56 (L2) and 89-97 (L3) (FIG. 1A). In the VH domain, positions 26-35(H1), 49-65 (H2) and 94-102 (H3) were grafted (FIG. 1B). In addition asecond HVR graft, Fib504Kgraft, was constructed that also includedwithin the HVR, VL position 49, based on an extended definition for L2(see MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)). MacCallum etal. have analyzed antibody and antigen complex crystal structures andfound position 49 of the light chain and positions 49 and 94 of theheavy chain are part of the antigen contact region thus, these positionswere included in the definitions of HVR-L2, HVR-H2 and HVR-H3 forhumanized anti-beta7 antibodies disclosed herein.

The direct-graft variants were generated by Kunkel mutagenesis (Kunkelet al. (1987) supra) using a separate oligonucleotide for eachhypervariable region. Correct clones were assessed by DNA sequencing.

Soft Randomization of the Hypervariable Regions:

The process of “soft randomization” (see U.S. Application Ser. No.60/545,840) refers a procedure for biased mutagensis of a selectedprotein sequence, such as a hypervariable region of an antibody. Themethod maintains a bias towards the murine, rat, or other startinghypervariable region sequence, while introducing an approximately 10-50percent mutation at each selected position. This technique increases thecapacity of the library screening employed and avoids a change in theantigen epitope recognized by the antibody. According to this softrandomization technique, sequence diversity is introduced into eachhypervariable region using a strategy that maintains a bias towards themurine hypervariable region sequence. This was accomplished using apoisoned oligonucleotide synthesis strategy first described by Gallop etal., J. Med. Chem. 37:1233-1251 (1994). However, other methods formaintaining a bias towards the non-human hypervariable region residueare available, such as error prone PCR, DNA shuffling, etc.

According to the method used herein, for a given position within ahypervariable region to be mutated, the codon encoding the wild-typeamino acid is poisoned with a mixture (e.g. a 70-10-10-10 mixture) ofnucleotides resulting in an approximately 50 percent mutation rate ateach selected hypervariable region position. To achieve this, the codonencoding the wild-type hypervariable region amino acid to be mutated issynthesized with a low level of contaminating mixture of the other threenucleotides, such as a 70-10-10-10 mixture of nucleotides. Thus, by wayof example, for soft randomization of PRO (CCG), the first positionsynthesized is a mixture of 70% C, and 10% each of G, T and A; thesecond position is a mixture of 70% C, and 10% each of A, G, and T; andthe third position is a mixture of 70% G, and 10% each of A, C and T. Itis understood that the bias can be adjusted up or down depending uponthe codon being synthesized at a given position, the number of codonsthat code for a particular amino acid, and the degree thatoligonucleotide synthesis is poisoned by the nucleotide composition ofthe synthesis mixture.

Soft randomized oligonucleotides can be patterned after the murine, rator other starting hypervariable region sequences and encompass the sameregions defined by the direct hypervariable region grafts. Optionally,two positions, amino acids at the beginning of H2 and H3 in the VHdomain, may be limited in their diversity: the codon RGC may be used forposition 49 encoding A, G, S or T and at position 94, the codon AKG maybe used encoding M or R.

Generation of phage libraries—Randomized oligonucleotide pools designedfor each hypervariable region were phoshorylated separately in six 20 μlreactions containing 660 ng of oligonucleotide, 50 mM Tris pH 7.5, 10 mMMgCl₂, 1 mM ATP, 20 mM DTT, and 5 U polynucleotide kinase for 1 h at 37°C. The six phosphorylated oligonucleotide pools were then combined with20 μg of Kunkel template in 50 mM Tris pH 7.5, 10 mM MgCl₂ in a finalvolume of 500 μl resulting in an oligonucleotide to template ratio of 3.The mixture was annealed at 90° C. for 4 min, 50° C. for 5 min and thencooled on ice. Excess, unannealed oligonucleotide was removed with aQIAQUICK™ PCR purification kit (Qiagen kit 28106, Qiagen, Valencia,Calif.) using a modified protocol to prevent excessive denaturation ofthe annealed DNA. To the 500 μl of annealed mixture, 150 μl of Qiagenbuffer PB was added, and the mixture was split between 2 silica columns.Following a wash of each column with 750 μl of Qiagen buffer PE and anextra spin to dry the columns, each column was eluted with 110 μl of 10mM Tris, 1 mM EDTA, pH 8. The annealed and cleaned-up template (220 μl)was then filled in by adding 1 μl 100 mM ATP, 10 μl 25 mM dNTPs (25 mMeach of dATP, dCTP, dGTP and dTTP), 15 μl 100 mM DTT, 25 μl 10× TMbuffer (0.5 M Tris pH 7.5, 0.1 M MgCl₂), 2400 U T4 ligase, and 30 U T7polymerase for 3 h at room temperature.

The filled-in product was analyzed on Tris-Acetate-EDTA/agarose gels(Sidhu et al., Methods in Enzymology 328:333-363 (2000)). Three bandsare usually visible: the bottom band is correctly filled and ligatedproduct, the middle band is filled but unligated and the top band isstrand displaced. The top band is produced by an intrinsic side activityof T7 polymerase and is difficult to avoid (Lechner et al., J. Biol.Chem. 258:11174-11184 (1983)); however, this band transforms 30-foldless efficiently than the bottom band and usually contributes little tothe library. The middle band is due to the absence of a 5′ phosphate forthe final ligation reaction; this band transforms efficiently and givesmainly wild type sequence.

The filled in product was then cleaned-up and electroporated into SS320cells and propagated in the presence of M13/KO7 helper phage asdescribed by Sidhu et al., Methods in Enzymology 328:333-363 (2000).Library sizes ranged from 1-2×10⁹ independent clones. Random clones fromthe initial libraries were sequenced to assess library quality.

Phage Selection—Full length human integrin alpha4beta7 was expressed in293 cells (Graham et al., J. Gen Virol. 36:59 (1977)), purified byFib504 affinity chromatography and used as the target for phageselection. For immobilization on MaxiSorp™ microtiter plates (NalgeNunc, Rochester, N.Y.), 100 μl of human integrin alpha4beta7 was coatedat 5 μg/ml in 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM CaCl₂, 2 mM MgCl₂ and2 mM MnCl₂ (TBSM), overnight at 4 degrees C. Wells were blocked for 1 husing TBSM containing 1% BSA. For the first round of selection, 8 wellscoated with target were used; a single target coated well was used forsuccessive rounds of selection. Phage were harvested from the culturesupernatant and suspended in TBSM containing 1% BSA and 0.05% TWEEN™ 20(TBSMBT). After binding to the wells for 2 h, unbound phage were removedby extensive washing with TBS containing 0.05 TWEEN 20 (TBST). Boundphage were eluted by incubating the wells with 100 mM HCl for 30 min.Phage were amplified using Top10 cells and M13/KO7 helper phage andgrown overnight at 37° C. in 2YT, 50 μg/ml carbanacillin. The titers ofphage eluted from a target coated well were compared to titers of phagerecovered from a non-target coated well to assess enrichment. After fourrounds of selection were performed, random clones were selected forsequence analysis.

Fab Production and Affinity Determination—To express Fab protein foraffinity measurements, a stop codon was introduced between the heavychain and g3 in the phage display vector. Clones were transformed intoE. coli 34B8 cells and grown in AP5 media at 30 C (Presta, L. et al.,Cancer Res. 57:4593-4599 (1997)). Cells were harvested bycentrifugation, suspended in 10 mM Tris, 1 mM EDTA pH 8 and broken openusing a microfluidizer. Fab was purified with Protein G affinityhromatography.

Affinity determinations were performed by surface plasmon resonanceusing a BIAcore™-3000 (Biacore, Piscataway, N.J.). Humanized Fib504 Fabvariants were immobilized in 10 mM acetate pH 4.5 (ranging from 250 to1500 response units (RU)) on a CMS sensor chip and 2-fold dilutions ofhuman integrin alpha4beta7 (1.5 to 770 nM) in TBSM containing 2%n-octylglucoside were injected. Each sample was analyzed with 5-minuteassociation and 5 to 60-minute dissociation times. After each injection,the chip was regenerated using three 1-minute injections of 8 M urea.Binding response was corrected by subtracting the RU from a blank flowcell. A 1:1 Languir model of simultaneous fitting of k_(on) and k_(off)was used for kinetics analysis.

Results and Discussion

Humanization of rat Fib504—The human acceptor framework used forhumanization is based on the framework used for HERCEPTIN® and consistsof the consensus human kappa I (huKI) VL domain and a variant of thehuman subgroup III (humIII) consensus VH domain. This variant VH domainhas 3 changes from the human consensus: R71A, N73T and L78A. The VL andVH domains of rat Fib504 were each aligned with the human kappa I andsubgroup III domains; each hypervariable region (HVR) was identified andgrafted into the human acceptor framework to generate a HVR graft (504graft) that could be displayed as an Fab on phage (FIGS. 1A and 1B).

Based on the analysis of available antibody and antigen complex crystalstructures MacCallum et al. (MacCallum et al. J. Mol. Biol. 262: 732-745(1996)) proposed HVR definitions based on variable domain residues thatfrequently contact antigens. Thus positions 49G and 94M of the heavychain were included in the HVR graft of Fib504 (FIG. 1B). In addition, asecond HVR graft, Fib504Kgraft, was also generated which includedposition 49K of the light chain, because this position is also withinthe contact definition of HVR-L2 and can serve as an antigen contact(FIG. 1A). When either the Fib504 or Fib504K grafts were displayed onphage and tested for binding to immobilized alpha4beta7, no binding wasobserved.

Libraries were generated using both the Fib504 and Fib504-K HVR graftsin which each of the HVR regions were soft randomized simultaneously.Each HVR graft library was panned against immobilized alpha4beta7 for 4rounds of selection. No enrichment was observed and clones picked forDNA sequence analysis displayed only random sequence changes targeted tothe 6 HVR regions.

Two additional VH framework sequences, “RL” and “RF” were investigatedas acceptor frameworks and contained changes at positions 71 and 78.Position 71 was changed to an Arginine as in the human subgroup IIIconsensus, and position 78 was changed to a Leucine as in the humansubgroup III consensus (acceptor framework “RL”) or a Phenylalanine asin the human subgroup II consensus and the rat Fib504 VH framework(acceptor framework “RF”) (FIG. 10A). When either the Fib504 or Fib504Kgraft in the “RL” (Fib504-RL and Fib504K-RL) or “RF” (Fib504-RF andFib504K-RF) acceptor framework was displayed on phage and tested forbinding to immobilized alpha4beta7. Specific phage binding was onlyobserved for the Fib504K graft using the “RF” framework (FIG. 10B). Themodest binding of phage displaying the Fib504-RF graft relative to theother grafts lacking Y49K (light chain) and L78F (heavy chain) indicatesthe importance of these positions in selecting a useful acceptorframework.

Libraries were generated as before using a soft randomization strategysimultaneously at each of the 6 HVRs for the Fib504K-RL and Fib504K-RFgrafts and sorted on immobilized alpha4beta7 for 4 rounds of selectionas described above. Enrichment was only observed for the library basedon the Fib504K-RF graft. Clones from round 4 of the Fib504K-RF librarywere selected for sequence analysis and revealed amino acid changestargeted to HVR-L1. Most clones contained the change Y32L; in additionposition 31 was frequently changed to D, S, P or N (FIG. 1C). Inaddition to the starting graft, Fib504K-RF, 3 clones were expressed andpurified as Fab protein and further analyzed by Biacore as describedabove. Clones hu504-5, hu504-16 and hu504-32 (variants of SEQ ID NO:1containing substituions T31S plus Y32L (variant hu504.5), Y32L (varianthu504.16), or T31D plus Y32L (variant hu504.32); see FIG. 1C), showedexcellent binding to alpha4beta7 relative to the Fib504K-RF graft andmet or exceeded the affinity of the chimeric Fib504 Fab for binding toalpha4beta7. The results of the Biacore analysis are shown in Table 3,below, and indicate that selected variation in the HVRs and/or frameworkregions, disclosed herein, generated antagonist antibodies toalpha4beta7 having improved affinity relative to the starting antibody.The results in Table 3 indicate that humanized variant 504.32 showed thegreatest increase in affinity relative to the starting rat antibody bybinding 3-fold more tightly to alpha4beta7.

TABLE 3 Fab Affinity to (BIAcore ™ analysis) Alpha4beta7 (nM) Fib504 11Variant 504.5 9 Variant 504.16 23 Variant 504.32 3

The results in Table 3 also indicate that the redesign of HVR-L1 wasimportant to the restoration of high affinity antigen binding. Inparticular, the mutation Y32L was most frequent among the variousclones. Other changes at position 31 and numerous other substitutionsthroughout HVR-H1 appear to be well tolerated and may provide additionalimprovement. From these results it is clear that there are multiplesequence changes that can improve the affinity of Fib504 grafted onto ahuman framework to generate affinities that meet or exceed that of theinitial rat antibody.

Thus, starting from the graft of the 6 rat Fib504 HVRs into the humanacceptor scaffold, the expansion of HVR-L2 to include position 49(Lysine), expansion of HVR-H2 to include position 49 (Glycine), and theexpansion of HVR-94 to include position 94 (Methionine) as well as aminoacid changes at position 32 of VHR-L1 (where L or I replace Y) and,optionally, at position 31 of the VHR-L1 (where T is replaced by D or S,for example). Useful framework amino acid changes were made at positions71 (A71R) and 78 (L78F) in the VH domain. Such amino acid changes leadto a fully human antibody, variant hu504.32, for example, with 3-foldimproved binding affinity for alpha4beta7 integrin. Furthermore,selected humanized antibodies described herein have been determined tohave at least comparable biological activity as the parent rat Fib504antibody (see Example 3 herein).

Example 2 Additional Humanized Fib504 HVR Variants

The HVR amino acid sequences of humanized variant Fib504.32 were furthermodified to generated additional variants capable of antagonizing theactivity of beta7 integrin subunit and/or integrins containing the beta7subunit.

Generating a broad amino acid scan library—A library to scan selectedHVR positions for other amino acid residues capable of generatingbeta7-binding variants of variant hu504.32 was generated using 3oligonucleotides: 504-L1, designed to soft randomize a portion of HVR-L1with a bias towards the hu504.32 HVR-L1 sequence (i.e. the sequenceASESVDDLLH (SEQ ID NO:47, for relative positions A2-A11) was softrandomized as described above); and HVR-L3 504-N96 and HVR-H3 504-M94which introduce NNS at positions HVR-L3 position 96 in the light chainand HVR-H3 position 94 in the heavy chain, thus allowing all 20 aminoacids at these positions. With these three oligonucleotides, the broadamino acid scan library was generated as described above using atemplate containing three stop codons in the light chain (positions 31and 32 in HVR-L1 and position 96 in HVR-L3) and 1 stop codon in theheavy chain (position 94 in HVR-H3). Broad amino acid scan ofhu504-32—To more fully explore the preferred sequences allowed in HVR-L1and to enhance the stability of 504-32, we designed a phage library thata) soft randomized HVR-L1 of 504-32 in the region where changes wereobserved (i.e. ASESVDDLLH (SEQ ID NO:47, for relative positions A2-A11)during humanization (FIG. 1C), and b) allowed all possible amino acidsat N96 in HVR-L3 and M94 in HVR-H3. Following 4 rounds of selectionagainst immobilized full-length human integrin alpha4beta7 as describedabove, 96 random clones were selected for sequence analysis. Thefrequency at which amino acids were found at each position in the broadamino acid scan library suggest that the HVR-L1 sequence present inhu504-32 and the methionine at position 94 in the heavy chain areoptimum for high affinity binding (FIG. 12). The most preferred aminoacids obtained by the selections starting from variant 504.32 (FIG. 12)are shown in yellow. In contrast, although asparagine is present atposition 96 in the light chain of hu504-32, the high frequency ofleucine observed at this position in the broad amino acid scan suggeststhe mutation N96L could further improve the affinity of humanized Fib504variants for alpha4beta7and also eliminate any potential deamidationproblems at this position. The information in FIG. 12 also suggests thata number of replacement amino acids are likely to be tolerated at mostpositions without a substantial loss in affinity. For example, toeliminate oxidation of M94 in HVR-H3, glutamine or arginine could likelybe substituted.

Generating the limited amino acid scan libraries—Six libraries for alimited amino acid scan utilized six different Kunkel templates, eachcontaining one stop codon located within one of the six HVRs. Eachlibrary was generated using a single oligonucleotide encoding a singleHVR and utilizing the codons listed in FIG. 11A (“codon” column) toalter amino acid residues for subsequent testing for binding to beta7 oralpha4beta7. The same procedures are used to alter amino acid residuesof anti-beta7 antibodies and test them for binding to alphaEbeta7integrins.

Limited amino acid scan of hu504-32—The limited amino acid scan ofhu504-16 was designed to make hu504-16 even more like the human lightand heavy chain consensus sequence and in the process identify theminimal sequence elements of rat Fib504 that are required for binding.Six libraries were generated and targeted at each HVR at positions thatdiffered between the hu504-16 and human consensus kappa I light orsubgroup III heavy chains (FIGS. 1A and 1B); either the rat or humanamino acid was allowed at these positions in the library (FIG. 11A). Inorder to accommodate coding for both amino acids during theoligonucleotide synthesis and mutagenesis, additional amino acids werealso introduced in some cases (see encoded amino acids, FIG. 11A). Thelimited amino acid scan libraries were selected against immobilizedfull-length human integrin alpha4beta7as described above andapproximately 32 random clones were sequenced from each library afterround 3. The frequency of each amino acid found at each position isshown in FIGS. 11B and 11C.

Like the broad amino acid scan, the limited amino acid scan alsoprovides information about what changes are tolerated at many positionsin humanized Fib504. Unlike the broad amino acid scan, however, thediversity allowed at each position randomized in the limited amino acidscan was restricted to a couple of amino acids. Thus the lack of anyobserved substitution at a given position does not indicate that aparticular residue can not be changed nor does the high frequency of anyparticular amino acid at a given position necessarily indicate that itis the best solution for high affinity.

At some positions (positions 27, 29, 30, 53, 54 of the light chain and50, 54, 58, 60, 61, and 65 of the heavy chain) the human consensus aminoacid is selected quite frequently suggesting a back mutation to thehuman consensus would not dramatically alter binding to humanalpha4beta7. In fact, at position 54 of the light chain (in HVR-L2), thehuman consensus amino acid is selected more frequently than the aminoacid from rat Fib504 indicating that this change made to 504-32 providesa useful beta7 binding antibody.

Further, as a result of the library design, amino acids that are notderived from either the human consensus or rat Fib504 are selected morefrequently at some positions and provide potential substitutions toimprove the affinity of humanized Fib504 variants. These include,without limitation, D30A and I55V in the light chain and Y50F in theheavy chain The results from these 2 libraries indicate that many HVRpositions tolerate other amino acid substitutions and still retaincomparable biological acitivity.

Summaries of observed amino acid changes are shown in FIGS. 13 and 15.FIG. 15 summarizes the various amino acids useful at each of thepositions in the CDRs of the antibody variants of the invention atpositions numbered according to Kabat numbering or a relative numberingsystem. Each of the additional antibodies encompassed by the variantsdepicted in FIGS. 13 and 15 is an embodiment of the invention.

Example 3 Cell Adhesion Assays

The ability of some of the humanized Fib504 variants of the invention tobind ligands expressed on a cell surface was tested by cell adhesionassays. Binding to alpha4beta7 and another beta7 integrin, alphaEbeta7were tested by the ability of the humanized variants to disrupt bindingof the integrin to its natural receptor. Binding of the humanized Fib504variants to beta7 subunit alone expressed on a cell surface wassimilarly tested. The procedures and the results are described below.

IgG production—Humanized Fib 504 IgG variants were expressed transientlyin 293 cells (Graham et al. (1977) supra) using a separate vector forthe light and heavy chains. The vectors were constructed by subcloningthe light or heavy variable domains into suitable expression vectors foreach of the light and heavy chains. Supernatant from 1.1 L CHO cellculture of a humanized Fib504 variant was filtered through a 0.45 umfilter and applied to a new 1 mL HiTrap Protein A HP column(Amersham/Pharmacia) equilibrated in Buffer A (10 mM tris pH 7.5, 150 mMNaCl). Samples were applied at 0.8 mL/min, overnight, 4 degrees C. Eachcolumn was then washed and equilibrated with 30 mL Buffer A. Elution ofantibody was accomplished by chromatography at room temperature on anFPLC (Amersham/Pharmacia) using a linear gradient of 14 min at 1 mL/minfrom 0 to 100% Buffer B (100 mM Glycine, pH 3.0). Resulting 1 mLfractions were immediately neutralized by the addition of 75 uL 1 Mtris, pH 8. Eluted protein was detected by absorption at 280 nm, andpeak fractions were pooled and desalted into PBS on PD10 G-25 sephadexdisposable sizing columns (Amersham/Pharmacia). Protein was detected byOD280 and peak fractions were pooled. The antibody in PBS was 0.22 umfiltered and stored at 4 degrees C. Amino acid analysis was used toquantify the concentrations of these purified antibodies, and valueswere assigned from the average of two separate determinations.

BCECF Labeling:

In each of the assays presented in this Example 3, cells were labeledaccording to the following procedure. All cells used in the adhesionassays were labeled with2′,7′-bis-(2-carboxylethyl)-5-(and-6)-carboxyfluorescein, acetoxymethylester (BCECF) at 10 uM in RPMI1640 media containing 10% FBS for RPMI8866cells and 38C13 cells transfected with beta7 subunit (38C13 beta7 cells)and in F-12:DMEM mix (50:50) containing 10% FBS foralphaEbeta7-transfected 293 cells (alphaEbeta7 293 cells). Cells werelabeled for 30 minutes and washed two times with assay media. Celldensity was adjusted to 3×10⁶ cells per ml for RPMI8866 and 38C13beta7cells and 2.2×10⁶ cells per ml for alphaEbeta7 293 cells.

Humanized Fib504 Variants Disrupt alpha4beta7 Binding to MAdCAM

RPMI8866/MAdCAM-1-Ig Cell Adhesion: RPMI8866 cells express alpha4beta7on their surface (Roswell Park Memorial Institute, Buffalo, N.Y.).Humanized Fib504 variants (hu504 variants) were contacted with a mixtureof RMPI8866 cells and MAdCAM fused to IgG coated on a solid support.Humanized Fib504 variant concentrations resulting in 50% inhibition(IC₅₀) of the binding of RPMI8866 cells to MAdCAM-1 were measured bycoating Nunc Maxisorp™ 96-well plates with 2 μg/ml in PBS, 100 μl/wellMAdCAM-1-Ig (Genentech, Inc., where Ig refers to fusion of MAdCAM-1 toan Fc region) overnight at 4° C. After blocking the plates with 200ul/well of 5 mg/ml BSA for one hour at room temperature, 50 μl ofhumanized Fib504 variants in assay media (RPMI 1640 media, Hyclone®,Logan Utah, USA, supplemented with 5 mg/mL BSA) were added to each welland 150,000 BCECF-labeled cells (BCECF, Molecular Probes, Eugene, Oreg.)in 50 μl of assay media were added to each well and incubated for 15minutes at 37° C. The wells were washed two times with 150 μl of assaymedia to remove unbound cells. The bound cells were solubilized with 100μl of 0.1% SDS in 50 mM Tris/HCl pH7.5. The amount of fluorescencereleased from lysed cells was measured by SPECTRAmax GEMINI™ (MolecularDevices, Sunnyvale, Calif.) at 485 nm excitation 530 nm emissionwavelengths. The fluorescence values were analyzed as a function of theconcentrations of the humanized Fib504 variants added in each assay,using a four-parameter nonlinear least squares fit, to obtain the IC₅₀values of each humanized Fib504 variant in the assay. IC₅₀ and IC₉₀values were estimated from the four-parameter fit. FIG. 14 is anexemplary plot of the results. The IC₉₀ and IC₅₀ values for each of thevariants tested are shown below in Table 4.

TABLE 4 Antibody binding to human MAdCAM-1 Antibody Tested: IC₅₀ (nM)IC₉₀ (nM) Fib504 and hu504 variants Exp 1/Exp 2* Exp 1/Exp 2* Rat Fib5040.098/0.197 0.483/0.703 Variant hu504.5 0.067/0.248 0.361/0.880 Varianthu504.16 0.0768/0.206  0.244/0.551 Variant hu504.32 0.036/0.1190.150/0.396 6B11 (non-blocking control) >100 >100 *Exp 1/Exp 2 refers tothe results of repeated assays.Humanized Fib504 Variant Disruption of alpha4beta7 Binding to VCAM

RPMI8866/7dVCAM-1 Cell Adhesion: The RPMI8866/7dVCAM-1 assay is similarformat to the RPMI8866/MAdCAM-1-Ig except that 7dVCAM-1 (ADP5, R&DSystems, Minneapolis, Minn.) was used at 2 ug/ml to coat plates. Theresults were analyzed as described above for the MAdCAM bindingexperiments. The IC₅₀ values for each of the variants tested are shownbelow in Table 5.

TABLE 5 Antibody binding to human VCAM Antibody Tested: IC₅₀ (nM) IC₉₀(nM) Fib504 and hu504 variants Exp 1/Exp 2* Exp 1/Exp 2* Rat Fib5040.107/0.193 0.396/0.580 Variant hu504.5 0.088/0.270 0.396/0.726 Varianthu504.16 0.098/0.223 0.261/0.774 Variant hu504.32 0.059/0.1100.183/0.337 6B11 (non-blocking control) >100 >100 *Exp 1/Exp 2 refers tothe results of repeated assays.Humanized Fib504 Variant Disruption of alphaEbeta7 Binding to HumanE-cadherin

AlphaEbeta7 293/huE-Cadherin Cell Adhesion: 293 cells (Graham et al.(1977) supra) were transfected with alphaE and beta7 (Genentech, Inc.).The assay format is similar to the RPMI8866/MAdCAM-1-Ig assay exceptthat the huE-Cadherin (648-EC, R&D Systems, Minneapolis, Minn.) was usedat 2 μg/ml to coat the plates. Plates were then blocked with 5 mg/ml BSAas mentioned above and 50 μl of FIB504 variants in assay media(F-12:DMEM (50:50) supplemented with 5 mg/ml BSA) were add to each welland 110,000 BCECF labeled cells in 50 ul of assay media were added toeach well and incubated for 15 minutes at 37° C. The wells were washedtwo times with 150 μl of assay media and the amount of fluorescencereleased by lysed cells was measured and analyzed as described above.Assay results from three experiments are shown in Table 6.

TABLE 6 Antibody binding to human E-Cadherin Antibody Tested: Fib504 andhu504 variants IC₅₀ (nM) IC₉₀ (nM) Rat Fib504 2.047/7.89/4.198.80/24.5/9.95 Variant hu504.5 2.132/10.18/4.77 7.99/28.7/10.19 Varianthu504.16 1.957/10.05/4.58 7.03/33.7/13.51 Variant hu504.321.814/6.99/3.47 8.8/24.5/11.73 HP2/1 (anti-alpha4,control) >100/>100/>100 >100/>100/>100Humanized Fib504 Variant Disruption of beta7 Binding to MAdCAM

38C13beta7/muMAdCAM-1-Ig Cell Adhesion Assay: The38C13beta7/muMAdCAM-1-Ig assay was similar format to theRPMI8866/MAdCAM-1-Ig except that muMAdCAM-1-Ig (Genentech, Inc.) wasused at 2 μg/ml to coat plates. 38C13 alpha4+ murine lymphoma cells(Crowe, D. T. et al., J. Biol. Chem. 269:14411-14418 (1994)) weretransfected with DNA encoding integrin beta7 such that alpha4beta7 wasexpressed on the cell surface. The ability of the antibodies variants todisrupt interaction between the cell membrane associated alpha4beta7 andMAdCAM was performed as above. Assay results are shown in Table 7. Assayresults are shown in Table 7. (IC50 and IC90 values for 2 experimentsare shown).

TABLE 7 Activity of hu504 variant antibodies in 38C13-beta7 expressingcells Binding to murine MAdCAM Antibody Tested: Fib504 and hu504variants IC₅₀ (nM) IC₉₀ (nM) Rat Fib504 0.682/0.306 2.869/1.51 Varianthu504.5 0.8587/0.466  2.322/2.61 Variant hu504.16 0.998/0.610 3.717/4.08Variant hu504.32 0.718/0.458  4.08/1.51Humanized Fib504 Variant Disruption of beta7 Binding to Murine VCAM

38C13beta7/muVCAM-1-Ig Cell Adhesion Assay: The 38C13beta7/muVCAM-1-Igassay was performed according to the murine MAdCAM-1-Ig/RPMI8866 cellbinding assay above, except that muVCAM-1-Ig (Genentech, Inc.) was usedat 2 μg/ml to coat plates. Results of the assay are shown in Table 8.(IC50 and IC90 values for 2 experiments are shown).

TABLE 8 Activity of hu504 variant antibodies in 38C13-beta7 expressingcells Binding to murine VCAM-1-Ig Antibody Tested: Fib504 and hu504variants IC₅₀ (nM) IC₉₀ (nM) Rat Fib504 0.845/0.447 2.903/2.30 Varianthu504.5 0.763/0.407 3.074/2.30 Variant hu504.16 0.835/0.584 2.857/1.84Variant hu504.32 0.562/0.330 2.004/1.84

The results of the humanized Fib504 variant binding studies demonstratethat the humanized antibody of the invention binds its target beta7integrin subunit as well as the alpha4beta7 and alphaEbeta7 integrinwith about the affinity of the starting rat antibody and, in someembodiments, with greater affinity. Thus, a humanized anti-beta7antibody according to the invention has uses in anti-beta7 integrintherapies, particularly human therapies.

Relative Activity of hu504.32 Variants of the Invention

Different amino acid variants of the hu504.32 antibody were tested inhuman and mouse cell adhesion assay for their ability to inhibitbeta7-containing receptor binding to its ligand according to the celladhesion assay methods disclosed herein. The RPMI8866/MAdCAM-1-Fc assaywas performed as described herein above. The alphaEbeta7-293/huE-cadherein assay was modified by the use of human E-cadherin-Fc as theligand (human E-cadherin-Fc, 648-EC, R&D Systems, Minneapolis, Minn.).The relative ability of hu504.32 variants to inhibit interaction ofhuman fibronectin (huFN40) with human alpha4beta7 receptor on PRMI8866cells was also examined. The RPMI8866/hu Fibronectin (huFN40) assay usedfor these studies was similar in format to the RPMI8866/MAdCAM-1-Igassay disclosed herein except that human fibronectin alpha-chymotrypticfragment 40 kDa (F1903, Chemicon International, Temecula, Calif.) wasused at 2 μg/ml to coat plates.

The ability of the hu504.32 variants to inhibit interaction of murinebeta7-containing receptors with murine MAdCAM-1 or murine VCAM-1 wasexamined. Murine MAdCAM-1-Fc and murine VCAM-1-Fc were inhibited frominteracting with murine lymphoma alpha4+ cells expressing murine beta7(38C13beta7 cells) by the hu504.32 variants. The murine MAdCAM-1-Fc andVCAM-1-Fc cell adhesion assays were performed similarly to thosedescribed herein above for human MAdCAM and VCAM. Where ligands werefused to Fc regions, the Fc receptors on the cells were blocked with 0.5anti-CD16/32 antibody (anti-Fcgamma III/II receptor antibody, catalogNo. 553142, BD Biosciences, San Jose, Calif.) per 1 million cells for 5minutes at room temperature. 150,000 labeled cells in 50 μl of assaymedium were added to each well and incubated for 13 minutes at 37° C.The wells were washed and the amount of fluorescence released from lysedcells was measured as disclosed herein above. The control antibody forthe human cell adhesion assays was the mouse monoclonal antibody tohuman serum albumin, 6B11 (Catalog No. ab10244, Novus Biologicals,Littleton, Colo., USA). The control antibody for the murine celladhesion assays was the rat anti-mouse integrin beta7 antibody, M293 (BDBiosciences, San Jose, Calif.), which does not compete with ligand orwith Fib504 for binding to integrin beta7.

The results of triplicate assays for the human and murine cell adhesionassays are provided in Tables 9 and 10, respectively.

TABLE 9 Activity of hu504.32 Variant Antibodies in Human Cell AdhesionAssays IC50 Ave ± SD RPMI8866/ αEβ7-293/ Antibody huMAdCAM-1- RPMI8866/huE-Cadherin- RPMI8866/ Variant Fc hu7dVCAM-1 Fc huFN40 hu504.32 0.088 ±0.035 0.101 ± 0.021 3.970 ± 1.664 0.100 ± 0.046 hu504.32M94Q 0.090 ±0.045 0.111 ± 0.035 4.130 ± 1.212 0.124 ± 0.056 hu504.32M94R 0.075 ±0.034 0.089 ± 0.009 3.963 ± 1.776 0.119 ± 0.056 Control(6B11) >100 >100 >100 >100

TABLE 10 Activity of hu504.32 Variant Antibodies in Murine Cell AdhesionAssays IC50 Ave ± SD 38C13beta7/ 38C13beta7/ Antibody VariantmuMAdCAM-1-Fc mu7dVCAM-1-Fc hu504.32 0.270 ± 0.041 0.228 ± 0.065hu504.32M94Q 0.370 ± 0.102 0.264 ± 0.083 hu504.32M94R 0.391 ± 0.1120.228 ± 0.081 Control (M293) >100 >100

The hu504.32 antibody has a methionine at position 94 of the heavy chainCDR3. The variants M94Q (or hu504.32Q) and M94R (or hu504.32R) haveglutamine or arginine, respectively, at position 94 of the hu504.32antibody variant. The hu504.32M, Q, and R antibodies substantiallyreduced integrin beta7 receptor-ligand interaction in in each of theassays and are, thus, potent inhibitors of beta7-mediated cell adhesion.

Antibody hu504.32R Activity In Vivo

The hu504.32R antibody variant was tested in vivo for its ability toreduce integrin beta7 receptor-ligand interaction and reduce lymphocyterecruitment to inflamed colon in an in vivo murine inflammatory boweldisease model. BALB/c mice and CB17 SCID mice were obtained from CharlesRiver Laboratories International, Inc. (Wilmington, Mass., USA).CD4⁺CD45Rb high T cell reconsituted SCID colitic mice were prepared byisolating CD4⁺CD45Rb high T cells from donor BALB/c mice andtransferring 3×10⁵ cells in 100 μl PBS intravenously. Control SCID micedid not receive CD4⁺CD45Rb high T cells. Reconstituted CD4+ mice meetingthe treatment group enrollment criteria of 10% weight loss from baselineor 15% from peak weight at week 4 were considered to have inducedinflammatory bowel disease and were selected for treatment.

On the day of treatment with test antibodies, donor BALB/c micemesenteric lymph node (MLN) cells were harvested and radiolabelled withCr⁵¹. Treatment involved prior administration of anti-GP120 antibody,hu504.32 anti-beta7 antibody, hu504.32R anti-beta7 antibody, or noantibody (control) intravenously, 200 μg/100 μl PBS. Thirty minutesafter antibody administration, Cr⁵¹-labelled MLN cells were injected,4×10⁶ cells/100 μl. One hour post-injection of labelled cells, mice wereeuthanized and spleen, colon, and peyers patch were collected, weighed,and total Cr⁵¹ radioactivity per organ was determined. FIG. 16 is a bargraph of the results of these tests showing the relative ability of theantibodies to block homing of radiolabelled T cells to the colon of miceexperiencing inflammatory bowel disease. Homing of T cells to inflamedcolon was inhibited by hu504.32 and hu504.32R anti-beta7 antibodiesrelative to negative control, anti-GP120 antibody. Distribution tospleen was similar for all of the antibodies. Thus, the hu504.32 andhu504.32R anti-beta7 antibodies effectively inhibit homing of T cells toinflamed colon in vivo.

Antibody glycation does not affect the ability of hu504.32R variant toblock MAdCAM-1 binding to alpha4beta7 receptor.

Glycation, the non-enzymatic glycosylation of proteins, can affectantibody-ligand interactions (see, for example, Kennedy, D. M. et al.,Clin Exp Immunol. 98(2):245-51 (1994). Glycation of lysine at position49 of the 504.32R Glycation of the lysine at light chain position 49 ofthe hu504.32R variant (HVR-L2 relative position B1) was observed but hadno significant affect on the ability of the antibody variant to blockthe binding of MAdCAM-1 to alpha4beta7 receptor-expressing RPMI8866cells. Determination of glycation and glycation levels was performedusing standard elctrospray ionization-mass spectroscopy (ESI-MS) and byboronate affinity chromatography. Boronate affinity HPLC methods usefulto test for glycation are found at, for example, Quan C. P. et al.,Analytical Chemistry 71(20):4445-4454 (1999) and Li Y. C. et al., J.Chromatography A, 909:137-145 (2001). The cell adhesion assay wasperformed according to the RPMI8866/MAdCAM-1-Fc cell adhesion assaydisclosed herein.

In alternative embodiments of the invention, glycation at position 49 isreduced or eliminated where position 49 comprises an amino acid otherthan lysine. The polypeptide or antibody of the invention encompasses asan amino acid at position 49 (HVR-L2 relative position B1) any of aminoacids A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y, whereeach letter refers to a amino acid according to the standardsingle-letter amino acid designation. Alternatively, the amino acid atposition 49 of the light chain of a 504.32R variant (or other 504variant) is selected from the group consisting of R, N, V, A, F, Q, H,P, I, or L. An amino acid useful at position 49 is selected, forexample, by displaying (preparing a phage library of) the hu504.32R Fabon phage (variant) and substituting separately, at the codon forposition 49, a codon for each of the 20 naturally occurring amino acids.Phage expressing the hu504.32R variants altered at position 49 aretested for binding to integrin beta7 and/or to a receptor comprisingintegrin beta7, such as alpha4beta7 or alphaEbeta7 receptors. Thosevariants which bind to beta7 integrin or the alpha4beta7 or alphaEbeta7receptors are further screened for the ability to inhibit integrin beta7receptor-ligand binding and in vivo efficacy as described herein.Alternative, naturally or non-naturally occurring amino acids may besubstituted at position 49 by standard mutagenesis techniques and testedin the cell adhesion and in vivo assays described herein. Alternatively,the amino acid at position 49 of the light chain is an amino acid otherthan lysine (K), and amino acids at any other HVR or framework positionor positions in the light chain and/or heavy chain is altered to selectfor a variant anti-beta7 binding polypeptide or antibody that exhibitsbinding affinity, in vitro and in vivo biological activity,pharmacokinetics, drug clearance and immunogenicity useful for reductionof inflammation by reducing the biological activity of integrin beta7.Mutagenesis and selection of such a polypeptide or antibody variant isperformed as disclosed herein and according to other standardtechniques. Such a variant anti-beta7 binding polypeptide or antibodyexhibits integrin beta7 binding affinity within 10,000-fold, 1000-fold,alternatively within 100-fold, alternatively within 10-fold,alternatively within 5-fold, alternatively within 2-fold of the bindingaffinity exhibited by the any of the humanized Fib504 variants disclosedherein.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

What is claimed is: 1-53. (canceled)
 54. An anti-beta7 antibodycomprising: an HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3,wherein each, in order, comprises RASESVDDLLH (SEQ ID NO:9), KYASQSIS(SEQ ID NO:2), QQGNSLPNT (SEQ ID NO:3), GFFITNNYWG (SEQ ID NO:4),GYISYSGSTSYNPSLKS (SEQ ID NO:5), and RTGSSGYFDF (SEQ ID NO:66). 55-60.(canceled)
 61. A method of inhibiting the interaction of a human beta7integrin subunit with a second integrin subunit and/or a ligand bycontacting a humanized anti-beta7 antibody with the beta7 integrin. 62.The method of claim 61, wherein the anti-beta7 antibody comprises anHVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each, inorder, comprises RASESVDDLLH (SEQ ID NO:9), KYASQSIS (SEQ ID NO:2),QQGNSLPNT (SEQ ID NO:3), GFFITNNYWG (SEQ ID NO:4), GYISYSGSTSYNPSLKS(SEQ ID NO:5), and RTGSSGYFDF (SEQ ID NO:66).
 63. A method of modulatingbeta7 integrin-mediated cellular adhesion and/or recruitment in a humanexperiencing a disorder by administering to the mammal an effectiveamount of a composition comprising a humanized anti-beta7 antibody and apharmaceutical carrier.
 64. The method of claim 63, wherein theanti-beta7 antibody comprises an HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2and HVR-H3, wherein each, in order, comprises RASESVDDLLH (SEQ ID NO:9),KYASQSIS (SEQ ID NO:2), QQGNSLPNT (SEQ ID NO:3), GFFITNNYWG (SEQ IDNO:4), GYISYSGSTSYNPSLKS (SEQ ID NO:5), and RTGSSGYFDF (SEQ ID NO:66).